Compounds, compositions and method for coloring edible materials

ABSTRACT

The present invention provides compounds isolated from avocado seeds for use as a natural colorant in edible materials. The compounds of the invention are useful for coloring edible materials red, orange or yellow. The invention also provides compositions and methods for coloring edible materials to a desired color such as red, orange or yellow.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/343,810 (now U.S. Pat. No. 11,001,601), filed on Nov. 4, 2016, whichclaimed benefit of priority to U.S. Provisional Application No.62/250,684, filed Nov. 4, 2015, which is hereby incorporated byreference in its entirety herein.

REFERENCE TO GOVERNMENT GRANT

This invention was made with government support under Grant No.PEN04565, awarded by The United States Department of Agriculture HatchAct and under Grant No. AT004678, awarded by the National Institutes ofHealth. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The global natural and synthetic food color market is estimated to reachUS $ 2.3 billion by 2019, with North America dominating the market,followed closely by Europe. This figure reflects the ubiquitousapplication of added food colorants throughout the world. Though the useof added food colorants has continued to grow, consumers have becomeincreasingly concerned by the perceived negative health risks which maybe associated with artificial food colors. This change in consumerdesire can be seen by the change in the global food colors market, asnatural food colors have begun to dominate the market, increasing from54.9% in 2014 to a predicted 60% by 2020, with particular interest inthose compounds responsible for yellow, orange, red, and pink colors.Despite the fact that consumers are beginning to show preference fornatural food colors over those not found in nature, it cannot beoverstated that being of natural origin, i.e. being produced by a livingorganism, does not signify that the consumption of such compounds issafe.

In humans, food color is irrefutably linked to their perception of foodsafety and flavor (Garber et al., 2000, J Mark Theory Pract 8:59-72) andhas a direct connection with human's sensory perception of foods. Thiscan be both a help and a hindrance when it comes to marketing new foodproducts. While vibrant novel colors catch the attention of consumers,it tends to only be helpful in the case nondescript flavors (Garber etal., 2000, J Mark Theory Pract 8:59-72).

Artificial colorants are those pigments which have been fully discoveredand synthesized in the laboratory, and are not of natural origin.Artificial colorants are generally vibrant have continued to gainpopularity due to their increased stability under a variety of heat,light, time, and other storage conditions. Although there is a standardto what colors are allowable in food, the data on the long term effectsof these compounds is limited, as is knowledge as to what dose isactually consumed by individuals on a regular basis. Still, the use ofartificial colorants in the United States has increased 5 fold from 5mg/capita/day in 1950 to 68 mg/capita/day in 2012 (Stevens et al., 2014,T Clin Pediatr 53:133-40). Currently, the Food and Drug Administration's(FDA) website lists thirteen certified FD&C colors and lakes permanentlylisted for use in food.

A natural colorant can be defined as any pigment which is produced byany organism such as a plant, animal, fungi, or microorganism (Lunninget al., 2007, In Food Colorants: Chemical and Functional Properties p557). In its use in a food, a natural colorant can either be extractedfrom its natural source, such as in the case of safranal from saffron,or after discovery can be synthesized in a laboratory for use, as iscommonly done with β-carotene found in carrots. The general perceptionof consumers is that natural food colorants are innately safer thantheir artificial counterparts. It is true that many natural colorantsoffer a variety of health benefits mainly due to their antioxidantproperties. However, the dose of any compound to be consumed must alwaysbe taken into consideration.

Polyphenol oxidases (PPO) are enzymes (EC 1.14.18.1) found almostuniversally in all varieties of organisms including bacteria, insects,crustaceans, mammals, fungi, and plants (Mayer, 2006, Photochemistry:67:2318-31). They are divided into the two subclasses of tyrosinases andlaccases. PPO contributes to the production of the brown pigment melaninin mammals and in plants it is responsible for the browning which occurswhen the flesh of a fruit or vegetable is sliced or bruised in thepresence of oxygen.

Due to the increasing interest in natural colorants, there is now muchfocus turning to their production, and specifically PPO colorants. Oneparticular class of pigment compounds is benzotropolones.Benzotropolones are characterized by a seven-membered tropolone ringattached to a six-membered aromatic ring and have been found throughoutnature in mushrooms, black teas, Chinese sage, and Mesotaeniumberggrenii, an extremophyte living on glaciers (Manet et al., 2004, JAgric Food Chem 52:2455-61; Ginda et al., 1988, Tetrahedron 29:4603-6;Kerschensteiner et al., 2011, Tetrahedron 67:1536-9; Remias et al.,2012, FEMS Microbiol Ecol 79:638-48). Benzotropolones are generallyyellow, orange, red, or brown in color, although one instance of a “darksolid with green metallic luster” was observed in the case ofaurantricholine. Upon addition of base, aurantricholine changedirreversibly to green-black, while upon addition of acid it producedyellow compounds of undetermined structure (Kandaswami et al., 2007US20070178216). Benzotropolone-glycosides tend to have low solubility inorganic solvents and may only be easily dissolved in water, makingstructure elucidation complex. Another common property of some is thatthey may be unstable, even at low temperatures or upon standing inorganic solvents. Benzotropolones have been reported to have healthbeneficial properties due to their antioxidant and anti-obesity nature.For example, theaflavins, and their polymerized form, thearubigins, havebeen reported to aid in weight loss and metabolic syndrome due theirability to decrease appetite, reduce adipose tissue, increase metabolismand energy levels and protect and enhance lean body mass (Kandaswami etal., 2007, US20070178216; Cornelius et al., 2007, US20090098224).Theaflavins have also been shown to be useful in the treatment ofalcoholic liver diseases (Li et al, 2014, US20150094364). As the desirefor natural alternatives to artificial colorants continues to grow, moreresearch will be needed on the potential positive and negative healtheffects of these and other benzotropolones.

The avocado (Persea americana Mill. Lauraceae) is a large drupe and hasthe highest oil content of all fruits, with the possible exception ofthe olive fruit. The avocado seed represents up to 16% of the totalweight of the fruit, has a complex phytochemical profile and a longhistory of ethnobotanical use. Historically, colored exudate fromavocado seeds was used as indelible ink by the Conquistadors in the1500s. When crushed in air, avocado seeds develop a stable orangepigment (Dabas et al., 2011. J. Food Sci 76:C1335-41; Dabas, 2012, Ph.D.Thesis, The Pennsylvania State University). This development of colorwas dependent on the action of the enzyme polyphenol oxidase, indicatingthat the resulting pigment is a polyphenolic compound. Further studiesare needed to determine the identity of the compounds responsible forthe orange color, and their colorant characteristics in various systems.

Thus, there is a need in the art for novel natural colorants. Thepresent invention fulfills this need.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a compound of general formula (A):

In one embodiment, in general formula (A),

R¹ to R⁸ are each independently selected from the group consisting ofhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heterocyclyl, substituted heterocyclyl, heteroaryl,substituted heteroaryl, (C(R⁹R¹⁰))_(n), (C(R⁹R¹⁰))_(n)OR¹¹,(C(R⁹R¹⁰))_(n)(NR¹²)R¹¹, N(R⁹R¹⁰), and OR⁹, wherein any two of R¹ to R⁸are optionally joined to form a ring, wherein the ring is optionallysubstituted;

each occurrence R⁹ and R¹° is independently selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, aryl, substituted aryl, heterocyclyl, substitutedheterocyclyl, heteroaryl, and substituted heteroaryl, wherein R⁹ and R¹⁰are optionally joined to form a ring, wherein the ring is optionallysubstituted;

each occurrence R¹¹ is independently selected from the group consistingof hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heterocyclyl, substituted heterocyclyl, heteroaryl,substituted heteroaryl, a monosaccharide, a disaccharide, and apolysaccharide;

each occurrence R¹² is independently selected from the group consistingof hydrogen alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heterocyclyl, substituted heterocyclyl, heteroaryl,and substituted heteroaryl;

each occurrence of n is independently an integer from 0 to 10; and

X is selected from the group consisting of O, NH and S.

In one embodiment, R³ and R⁵ are joined to form a ring. In oneembodiment, R¹ is (C(R⁹R¹⁰))_(n)OR¹¹. In one embodiment, R¹¹ is amonosaccharide.

In one embodiment, the compound of general formula (A) is represented byformula (B)

In one embodiment, in general formula (B),

R² to R⁸ are each independently selected from the group consisting ofhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heterocyclyl, substituted heterocyclyl, heteroaryl,substituted heteroaryl, (C(R⁹R¹⁰))_(n), (C(R⁹R¹⁰))_(n)OR¹¹,(C(R⁹R¹⁰))_(n)(NR¹²)R¹¹, N(R⁹R¹⁰), and OR⁹, wherein any of R¹ to R⁸ areoptionally joined to form a ring, wherein the ring is optionallysubstituted;

each occurrence R⁹ and R¹⁰ is independently selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, aryl, substituted aryl, heterocyclyl, substitutedheterocyclyl, heteroaryl, and substituted heteroaryl, wherein R⁹ and R¹⁰are optionally joined to form a ring wherein the ring is optionallysubstituted;

each occurrence R¹¹ is independently selected from the group consistingof hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heterocyclyl, substituted heterocyclyl, heteroaryl,substituted heteroaryl, a monosaccharide, a disaccharide, and apolysaccharide;

each occurrence R¹² is independently selected from the group consistingof hydrogen alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heterocyclyl, substituted heterocyclyl, heteroaryl,and substituted heteroaryl;

each occurrence of n is independently an integer from 0 to 10;

m is an integer from 1 to 11;

p is an integer from 0 to 5; and

X is selected from the group consisting of O, NH and S.

In one embodiment, the compound of general formula (A) is represented bygeneral formula (C)

In one embodiment, in formula (C),

R¹, R², R⁴, and R⁶-R⁸ are each independently selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, aryl, substituted aryl, heterocyclyl, substitutedheterocyclyl, heteroaryl, substituted heteroaryl, (C(R⁹R¹⁰))_(n),(C(R⁹R¹⁰))_(n)OR¹¹, (C(R⁹R¹⁰))_(n)(NR¹²)R¹¹, N(R⁹R¹⁰), and OR⁹, whereinany of R¹, R², R⁴, and R⁶-R⁸ are optionally joined to form a ring,wherein the ring is optionally substituted;

each occurrence R⁹ and R¹⁰ is independently selected from the groupconsisting of hydrogen, an alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heterocyclyl,substituted heterocyclyl, heteroaryl, and substituted heteroaryl,wherein R⁹ and R¹⁰ are optionally joined to form a ring;

each occurrence R¹¹ is independently selected from the group consistingof hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heterocyclyl, substituted heterocyclyl, heteroaryl,substituted heteroaryl, a monosaccharide, a disaccharide, and apolysaccharide;

each occurrence R¹¹ is independently selected from the group consistingof hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heterocyclyl, substituted heterocyclyl, heteroaryl,and substituted heteroaryl;

each occurrence of n is independently an integer from 0 to 10;

X is selected from the group consisting of O, NH and S; and

A is an optionally substituted 3 to 10 membered ring.

In one embodiment, the compound is

In one embodiment, the compound is a hue selected from the groupconsisting of yellow, orange and red.

In another aspect, the invention provides an edible material comprisinga compound of the invention. In one embodiment, the edible material hasa hue selected from the group consisting of orange, red and yellow.

In another aspect, the invention provides a method of coloring an ediblematerial, the method comprising adding to the edible material a compoundof the invention.

In another aspect, the invention provides a compound prepared by aprocess comprising the steps of: obtaining a seed of Persea americana;grounding the seed to a slurry; incubating the powder; extracting thecompound by incubating the powder with an alcohol to form a firstmixture; isolating a first liquid from the first mixture; removing thestarch from the first liquid; precipitating an impurity in the liquid toform a second mixture; isolating a second liquid from the secondmixture; precipitating an insoluble material from the second mixture toform a third mixture; isolating a third liquid from the third mixture;adsorbing the third liquid to a resin; and isolating the compound byeluting the compound from the resin with an alcohol.

In one embodiment, the alcohol is methanol, ethanol, acetone, citricacid, acetic acid, or any combination thereof. In one embodiment, theresin is a XAD-7 resin.

In yet another aspect, the invention provides a method of imparting acolor to a substrate. In one embodiment the method comprises applying acompound of the invention to the substrate. In one embodiment, color isselected from the group consisting of red, yellow and orange. In oneembodiment, the substrate is an edible material

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1 depicts results of experimental examples demonstrating the colorof semi-pure colored avocado seed extract (CASE) in white grapefruitjuice, apple juice, and Sprite. Concentration of semi-pure CASE used isshown above each sample in the units of mg/mL.

FIG. 2 depicts results of experimental examples showing the ΔE values ofsemi-pure CASE in soda (Sprite), apple juice, and white grapefruitjuice.

FIG. 3A and FIG. 3B depict results of experimental examplesdemonstrating the color of semi-pure CASE in white cake. FIG. 3A depictsthe tops of the cupcakes. FIG. 3B depicts the middles of the cupcakes.Concentration of CASE is shown in mg/mL.

FIG. 4 depicts results of experimental examples showing the ΔE values ofsemi-pure CASE in cupcake tops and middles.

FIG. 5A and FIG. 5B depict results of experimental examplesdemonstrating the color of cheese when semi-pure CASE was added to awhite no-color-added cheese powder (blank). Warm milk was then added tothe resulting samples in order to prepare a cheese sauce. FIG. 5Adepicts the color of dry cheese powders. FIG. 5B depicts the color ofprepared cheese sauce.

FIG. 6 depicts results of experimental examples demonstrating the ΔEvalues of CASE in a white no-color-added Kraft cheese powder. ΔE of thewhite and regular Kraft cheese powders were also calculated and appearas zero-points along the x-axis.

FIG. 7A through FIG. 7D depict results of experimental examplesdemonstrating the change in ΔE of samples. FIG. 7A depicts the change oflighted samples at 26° C. FIG. 7B depicts the change of samples kept inthe dark at 4° C. FIG. 7C depicts the change of samples kept in the dark23° C. FIG. 7D depicts the change of samples kept in the dark 40° C.FIG. 8 depicts results of experimental examples demonstrating color ofCASE in model sugar drink samples on day 36 of the stability study.

FIG. 9A through FIG. 9D depict results of experimental examplesdemonstrating the change in 445 nm absorbance. FIG. 9A depicts thechange of lighted samples at 26° C. FIG. 9B depicts the change ofsamples kept in the dark at 4° C. FIG. 9C depicts the change of sampleskept in the dark 23° C. FIG. 9D depicts the change of samples kept inthe dark 40° C.

FIG. 10A through FIG. 10C depict results of experimental examplesdemonstrating semi-pure CASE samples compared to a control sample. FIG.10A depicts the color of pretreatment samples. FIG. 10B depicts thecolor of pH adjusted samples. FIG. 10C depicts the color of sampleswhere the pH returned to acidic conditions.

FIG. 11 depicts results of experimental examples demonstrating the fullLC profile of the semi-pure CASE in water control sample (top) and basetreated semi-pure CASE in water (bottom). The peak of interest, F12appears at 15 min on both chromatograms. Line colors are pink, 280 nm;blue, 320 nm; green, 445 nm.

FIG. 12 depicts results of experimental examples demonstrating the LCprofile and areas of maximum absorbance for F12 peak in semi-pure CASEin water (top) and pH adjusted semi-pure CASE in water. Line colors arepink, 280 nm; blue, 320 nm; green, 445 nm.

FIG. 13 depicts results of experimental examples demonstrating the PCAclustering scores for samples analyzed in positive mode.

FIG. 14 depicts results of experimental examples demonstrating the PCAof colored and uncolored extracts in positive mode.

FIG. 15 depicts results of experimental examples demonstrating the PCAof colored (solid line) and uncolored (dashed line) extracts in positivemode.

FIG. 16 depicts results of experimental examples demonstrating the PCAof colored and uncolored extracts in positive mode.

FIG. 17 depicts results of experimental examples of the HPLCchromatograph of the semi-pure, post-amberlite CASE. Samples wereanalyzed at 280 nm (top, black) and at 445 nm (bottom, red).

FIG. 18 depicts results of experimental examples of the HPLCchromatograph post-C18 rough F12. Samples were analyzed at 280 nm(bottom, black) and at 445 nm (top, red).

FIG. 19 depicts results of experimental examples demonstrating the MS/MSanalysis indicated a [M+H]⁺ 603.1675 parent peak.

FIG. 20 depicts results of experimental examples demonstrating analysisof pure F12 included a [M+H]⁺ 917.2639 peak (A), the compound ofinterest, [M+H]⁺ 603.1675 peak (B), a [M+H]⁺ 603.1687 peak (C), and[M+H]+ 1205 dimer produced from the combination of two [M+H]⁺ 603compounds (D).

FIG. 21 depicts results of experimental examples demonstrating theATR-FTIR analysis of “pure F12,” the most prominent colored compound.

FIG. 22 depicts results of experimental examples showing the structureof the most prominent colored compound, F12.

FIG. 23 depicts the ¹H NMR spectrum of F12 in (CD₃)₂SO.

FIG. 24 depicts the ¹³C NMR spectrum of F12 in (CD₃)₂SO.

FIG. 25 depicts the DEPT-edited HSQC spectrum of F12 in (CD₃)₂SO.

FIG. 26 depicts the HMBC NMR spectrum, of F12 in (CD₃)₂SO. Arrows on thestructure indicate correlations.

FIG. 27 depicts the COSY analysis of F12 in (CD₃)₂SO.

FIG. 28 depicts the TOCSY analysis of F12 in (CD₃)₂SO.

FIG. 29 depicts results of experimental examples demonstrating potentialprecursors for enzymatic synthesis of F12.

FIG. 30 depicts the ¹H NMR spectrum of F12 in D₂O.

FIG. 31 depicts the ¹C NMR spectrum of F12 in D₂O.

FIG. 32 depicts the DEPT-edited HSQC spectrum of F12 in D₂O.

FIG. 33 depicts the HMBC spectrum of F12 in D₂O.

FIG. 34 depicts the COSY analysis of F12 in D₂O.

FIG. 35 depicts the TOCSY analysis of F12 in D₂O.

FIG. 36 depicts the NOESY analysis of F12 in D₂O.

FIG. 37 depicts results of experimental examples demonstrating theeffect of semi-pure CASE on viability of LNCaP cells.

FIG. 38 depicts the chemical structures of F12 derivatives 1-10.

FIG. 39 depicts a diagram of the experimental protocol.

FIG. 40 depicts the absorbance spectrum of a sample.

FIG. 41 depicts the evolution of mean absorbance with temperature and pH(3 samples for each measure).

FIG. 42 depicts the stability of the mean absorbance for 3 daysdepending on the temperature, done on three seeds for each.

FIG. 43 depicts the difference of color of the same solution dependingon the pH.

FIG. 44 depicts a solution which was brought from pH 2 to pH 11 (left)and a control solution at pH 2 (right).

FIG. 45 depicts the evolution of the absorbance at 418 nm for a sampleat pH 11.

FIG. 46 depicts a titration curve of the solution with sodium hydroxide.

FIG. 47 depicts the difference of color depending on the composition infresh seeds of the solutions.

FIG. 48 depicts the difference of mean absorbance at the peak (470 nm)in fresh seeds of the solutions.

FIG. 49 depicts a cloudy and uncolored precipitate from a solution at pH11 in acidified ethanol.

FIG. 50 depicts small red balls of precipitate from a solution at pH 2in acidified ethanol.

FIG. 51 depicts the raw material, the avocado seed, immediately afterbeing cut and 15 minutes after being cut

FIG. 52 depicts a blended avocado seed sample before and afterfiltration.

FIG. 53 depicts the absorbance of basic avocado extract (BAE) at, pH10.58, 11.44 and 11.40.

FIG. 54 depicts the absorbance of basic avocado extract at pH 10.12 and11.24 and the absorbance measurement of 0.05% basic avocado extract attime zero and followed by every 30 mins for 5 hrs.

FIG. 55 depicts the precipitation of neutral avocado extract solutionbefore and after pipette mixing.

FIG. 56 depicts the absorbance of neutral avocado extract (NAE).

FIG. 57 depicts a comparison of base washed avocado extract unfilteredand filtered by the syringe.

FIG. 58 depicts a comparison between absorbance of normal, basic andbase washed avocado extracts.

FIG. 59 depicts the absorbance of different concentrations of AscorbicAcid in 0.25% Avocolor over 2 weeks.

FIG. 60 depicts the absorbance of Ascorbic Acid in 0.25% Avocolor over 2weeks.

FIG. 61 depicts the absorbance of different concentrations of AscorbicAcid in 0.25% Avocolor over 2 weeks.

FIG. 62 depicts the absorbance of Ascorbic Acid in 0.25% Avocolor over 2weeks.

FIG. 63 depicts the absorbance of different concentrations of Potassiummetabisulfite in 0.25% Avocolor over 2 weeks.

FIG. 64 depicts the absorbance of Potassium metabisulfite in 0.25%Avocolor over 2 weeks.

FIG. 65 depicts the absorbance of 0.1% Gelatin in 0.25% Avocado seedextract solution over 3 weeks.

FIG. 66 depicts the absorbance of 2% Casein in 0.25% Avocado seedextract solution over 3 weeks.

FIG. 67 depicts the absorbance of 0.2% Cherry flavoring in 0.25% Avocadoseed extract solution over 3 weeks.

FIG. 68 depicts the color of 2 mL of 1% Avocolor solution compared to0.2 mL of 1% Avocolor solution.

FIG. 69 depicts the color of 1% Avocolor solution in Sprite compared to1%Avocolor solution in deionized water.

FIG. 70 depicts the absorbance of different concentrations of Avocolorin Sprite over 2 days.

FIG. 71 depicts the absorbance of 1% Avocado seed extract solution inSprite in 2 days

FIG. 72 depicts the colors of different concentration of % Avocolorsolution in 10 mL of Sprite.

FIG. 73 depicts the colors of Maltodextrin Avocolor extract powder onCorn Chip.

FIG. 74 depicts the colors of of Maltodextrin Avocolor extract powder inwhite chocolate.

FIG. 75 depicts a flow chart demonstrating the method of isolatingperseoranjin.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to the unexpected identification of novelcompounds isolated from colored avocado seed extract and their utilityas source of natural colorants. In some aspects the compounds may beused as an orange colorant. In another embodiment, the compounds may beused as a yellow colorant. In yet another embodiment, the compounds maybe used as a red colorant. However, the invention should not be limitedto only these colors. Rather, the invention includes any desired colorthat is associated with one or more of hues yellow, orange, and red. Inone embodiment, the invention includes any color in the spectrum foryellow, orange, and red. In one embodiment, the invention includes anycolor that contains one or more of yellow, orange, and red.

Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures inbiochemistry, analytical chemistry and organic chemistry are thosewell-known and commonly employed in the art. Standard techniques ormodifications thereof are used for chemical syntheses and chemicalanalyses.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

As used herein, the term “benzotropolone” refers to a seven-memberedtropolone ring attached to a six-membered aromatic ring.

As used herein, the term colored avocado seed extract (CASE),perseoranjin, F12, and Avocolor are used to describe a composition forcoloring food which is isolated from an Avocado seed using a method ofthe invention. In one embodiment, CASE, perseoranjin, F12, and Avocolorcomprise a compound of the invention.

In one embodiment, compounds of the invention contain saccharides.“Saccharides” as used herein, include, but are not limited to aldose orketose pentosyl or hexosyl sugars selected from the group consisting ofD- and L-enantiomers of ribose, glucose, galactose, mannose, arabinose,allose, altrose, gulose, idose, talose and their substitutedderivatives. Most preferably, the subject sugar comprises an aldosepentosyl or hexosyl sugar selected from ribose, glucose, galactose,glucosamine, galactosamine, N-acetylglucosamine, N-acetylgalactosamine,N-acetyl ribosamine, xylose, mannose and arabinose.

“Di-saccharide”, when used in regard to the subject sugar residue, isintended to mean a polymeric assemblage of 2 sugar residues.Representative examples of disaccharides include homo-polymeric (e.g.,maltose and cellobiose) and hetero-polymeric (e.g., lactose and sucrose)assemblages of sugars as set forth supra.

“Tri-saccharide”, when used in regard to the subject sugar residue, isintended to mean a polymeric assemblage of 3 sugar residues.

“Polysaccharide”, when used in regard to the subject sugar residue, isintended to mean a polymeric assemblage of 3 or more sugar residues.

An “effective amount” of a delivery vehicle is that amount sufficient toeffectively bind or deliver a compound.

The term “compound,” as used herein, unless otherwise indicated, refersto any specific chemical compound disclosed herein. In one embodiment,the term also refers to stereoisomers and/or optical isomers (includingracemic mixtures) or enantiomerically enriched mixtures of disclosedcompounds.

As used herein, the term “alkyl,” by itself or as part of anothersubstituent means, unless otherwise stated, a straight or branched chainhydrocarbon having the number of carbon atoms designated (i.e. C₁₋₆means one to six carbon atoms) and including straight, branched chain,or cyclic substituent groups. Examples include methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, andcyclopropylmethyl. Most preferred is (C₁-C₆)alkyl, particularly ethyl,methyl, isopropyl, isobutyl, n-pentyl, n-hexyl and cyclopropylmethyl.

As used herein, the term “alkenyl,” employed alone or in combinationwith other terms, means, unless otherwise stated, a stablemono-unsaturated, di-unsaturated, or polyunsaturated straight chain orbranched chain hydrocarbon group having the stated number of carbonatoms. Examples include vinyl, propenyl (or allyl), crotyl, isopentenyl,butadienyl, 1,3-pentadienyl, 1,4-pentadienyl, and the higher homologsand isomers. A functional group representing an alkene may beexemplified by —CH₂—CH═CH₂.

As used herein, the term “alkynyl,” employed alone or in combinationwith other terms, means, unless otherwise stated, a stable straightchain or branched chain hydrocarbon group with a triple carbon-carbonbond, having the stated number of carbon atoms. Non-limiting examplesinclude ethynyl and propynyl, and the higher homologs and isomers. Theterm “propargylic” refers to a group exemplified by —CH₂—CCH. The term“homopropargylic” refers to a group exemplified by —CH₂CH₂—CCH. The term“substituted propargylic” refers to a group exemplified by —CR₂—CCR,wherein each occurrence of R is independently H, alkyl, substitutedalkyl, alkenyl or substituted alkenyl, with the proviso that at leastone R group is not hydrogen. The term “substituted homopropargylic”refers to a group exemplified by —CR₂CR₂—CCR, wherein each occurrence ofR is independently H, alkyl, substituted alkyl, alkenyl or substitutedalkenyl, with the proviso that at least one R group is not hydrogen.

As used herein, the term “substituted alkyl,” “substituted cycloalkyl,”“substituted alkenyl” or “substituted alkynyl” means alkyl cycloalkyl,alkenyl or alkynyl, as defined above, substituted by one, two or threesubstituents selected from the group consisting of halogen, —OH, alkoxy,—NH₂, —N(CH₃)₂, —C(═O), —C(═O)OH, trifluoromethyl, —C(═O)O(C₁-C₄)alkyl,—C(═O)NH₂, —SO₂NH₂, —C(═NH)NH₂, and —NO₂, preferably containing one ortwo substituents selected from halogen, —OH, alkoxy, —NH₂,trifluoromethyl, —N(CH₃)₂, and —C(═O)OH, more preferably selected fromhalogen, alkoxy and —OH. Examples of substituted alkyls include, but arenot limited to, 2,2-difluoropropyl, 2-carboxycyclopentyl and3-chloropropyl.

As used herein, the term “heteroalkyl” by itself or in combination withanother term means, unless otherwise stated, a stable straight orbranched chain alkyl group consisting of the stated number of carbonatoms and one or two heteroatoms selected from the group consisting ofO, N, and S, and wherein the nitrogen and sulfur atoms may be optionallyoxidized and the nitrogen heteroatom may be optionally quaternized. Theheteroatom(s) may be placed at any position of the heteroalkyl group,including between the rest of the heteroalkyl group and the fragment towhich it is attached, as well as attached to the most distal carbon atomin the heteroalkyl group. Examples include: —O—CH₂—CH₂—CH₃,—CH₂—CH₂—CH₂—OH, —CH₂—CH₂—NH—CH₃, —CH₂—S—CH₂—CH₃, and —CH₂CH₂—S(═O)—CH₃.Up to two heteroatoms may be consecutive, such as, for example,—CH₂—NH—OCH₃, or —CH₂—CH₂—S—S—CH₃

As used herein, the term “alkoxy” employed alone or in combination withother terms means, unless otherwise stated, an alkyl group having thedesignated number of carbon atoms, as defined above, connected to therest of the molecule via an oxygen atom, such as, for example, methoxy,ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homologs andisomers. Preferred are (C₁-C₃) alkoxy, particularly ethoxy and methoxy.

As used herein, the term “halo” or “halogen” alone or as part of anothersubstituent means, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom, preferably, fluorine, chlorine, or bromine,more preferably, fluorine or chlorine.

As used herein, the term “cycloalkyl” refers to a mono cyclic orpolycyclic non-aromatic radical, wherein each of the atoms forming thering (i.e. skeletal atoms) is a carbon atom. In one embodiment, thecycloalkyl group is saturated or partially unsaturated. In anotherembodiment, the cycloalkyl group is fused with an aromatic ring.Cycloalkyl groups include groups having from 3 to 10 ring atoms.Illustrative examples of cycloalkyl groups include, but are not limitedto, the following moieties:

Monocyclic cycloalkyls include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.Dicyclic cycloalkyls include, but are not limited to,tetrahydronaphthyl, indanyl, and tetrahydropentalene. Polycycliccycloalkyls include adamantine and norbornane. The term cycloalkylincludes “unsaturated nonaromatic carbocyclyl” or “nonaromaticunsaturated carbocyclyl” groups, both of which refer to a nonaromaticcarbocycle as defined herein, which contains at least one carbon carbondouble bond or one carbon carbon triple bond.

As used herein, the term “heterocycloalkyl” or “heterocyclyl” refers toa heteroalicyclic group containing one to four ring heteroatoms eachselected from O, S and N. In one embodiment, each heterocycloalkyl grouphas from 4 to 10 atoms in its ring system, with the proviso that thering of said group does not contain two adjacent O or S atoms. Inanother embodiment, the heterocycloalkyl group is fused with an aromaticring. In one embodiment, the nitrogen and sulfur heteroatoms may beoptionally oxidized, and the nitrogen atom may be optionallyquaternized. The heterocyclic system may be attached, unless otherwisestated, at any heteroatom or carbon atom that affords a stablestructure. A heterocycle may be aromatic or non-aromatic in nature. Inone embodiment, the heterocycle is a heteroaryl.

An example of a 3-membered heterocycloalkyl group includes, and is notlimited to, aziridine. Examples of 4-membered heterocycloalkyl groupsinclude, and are not limited to, azetidine and a beta lactam. Examplesof 5-membered heterocycloalkyl groups include, and are not limited to,pyrrolidine, oxazolidine and thiazolidinedione. Examples of 6-memberedheterocycloalkyl groups include, and are not limited to, piperidine,morpholine and piperazine. Other non-limiting examples ofheterocycloalkyl groups are:

Examples of non-aromatic heterocycles include monocyclic groups such asaziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine,pyrroline, pyrazolidine, imidazoline, dioxolane, sulfolane,2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane,piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine,morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran,1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane,4,7-dihydro-1,3-dioxepin, and hexamethyleneoxide.

As used herein, the term “aromatic” refers to a carbocycle orheterocycle with one or more polyunsaturated rings and having aromaticcharacter, i.e. having (4n+2) delocalized 7C (pi) electrons, where n isan integer.

As used herein, the term “aryl,” employed alone or in combination withother terms, means, unless otherwise stated, a carbocyclic aromaticsystem containing one or more rings (typically one, two or three rings),wherein such rings may be attached together in a pendent manner, such asa biphenyl, or may be fused, such as naphthalene. Examples of arylgroups include phenyl, anthracyl, and naphthyl. Preferred examples arephenyl and naphthyl, most preferred is phenyl.

As used herein, the term “aryl-(C₁-C₃)alkyl” means a functional groupwherein a one- to three-carbon alkylene chain is attached to an arylgroup, e.g., —CH₂CH₂-phenyl. Preferred is aryl-CH₂— and aryl-CH(CH₃)—.The term “substituted aryl-(C₁-C₃)alkyl” means an aryl-(C₁-C₃)alkylfunctional group in which the aryl group is substituted. Preferred issubstituted aryl(CH₂)—. Similarly, the term “heteroaryl-(C₁-C₃)alkyl”means a functional group wherein a one to three carbon alkylene chain isattached to a heteroaryl group, e.g., —CH₂CH₂-pyridyl. Preferred isheteroaryl-(CH₂)—. The term “substituted heteroaryl-(C₁-C₃)alkyl” meansa heteroaryl-(C₁-C₃)alkyl functional group in which the heteroaryl groupis substituted. Preferred is substituted heteroaryl-(CH₂)—.

As used herein, the term “heteroaryl” or “heteroaromatic” refers to aheterocycle having aromatic character. A polycyclic heteroaryl mayinclude one or more rings that are partially saturated. Examples includethe following moieties:

Examples of heteroaryl groups also include pyridyl, pyrazinyl,pyrimidinyl (particularly 2- and 4-pyrimidinyl), pyridazinyl, thienyl,furyl, pyrrolyl (particularly 2-pyrrolyl), imidazolyl, thiazolyl,oxazolyl, pyrazolyl (particularly 3- and 5-pyrazolyl), isothiazolyl,1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl,1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and1,3,4-oxadiazolyl.

Examples of polycyclic heterocycles and heteroaryls include indolyl(particularly 3-, 4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl,tetrahydroquinolyl, isoquinolyl (particularly 1- and 5-isoquinolyl),1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (particularly 2-and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl,1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl,benzofuryl (particularly 3-, 4-, 5-, 6- and 7-benzofuryl),2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (particularly3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl(particularly 2-benzothiazolyl and 5-benzothiazolyl), purinyl,benzimidazolyl (particularly 2-benzimidazolyl), benzotriazolyl,thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, andquinolizidinyl.

As used herein, the term “substituted” means that an atom or group ofatoms has replaced hydrogen as the substituent attached to anothergroup. The term “substituted” further refers to any level ofsubstitution, namely mono-, di-, tri-, tetra-, or penta-substitution,where such substitution is permitted. The substituents are independentlyselected, and substitution may be at any chemically accessible position.In one embodiment, the substituents vary in number between one and four.In another embodiment, the substituents vary in number between one andthree. In yet another embodiment, the substituents vary in numberbetween one and two.

As used herein, the term “optionally substituted” means that thereferenced group may be substituted or unsubstituted. In one embodiment,the referenced group is optionally substituted with zero substituents,i.e., the referenced group is unsubstituted. In another embodiment, thereferenced group is optionally substituted with one or more additionalgroup(s) individually and independently selected from groups describedherein.

In one embodiment, the substituents are independently selected from thegroup consisting of oxo, halogen, —CN, —NH₂, —OH, —NH(CH₃), —N(CH₃)₂,alkyl (including straight chain, branched and/or unsaturated alkyl),substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, fluoro alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted alkoxy, fluoroalkoxy,—S-alkyl, S(═O)₂alkyl, —C(═O)NH[substituted or unsubstituted alkyl, orsubstituted or unsubstituted phenyl], —C(═O)N[H or alkyl]₂,—OC(═O)N[substituted or unsubstituted alkyl]₂, —NHC(═O)NH[substituted orunsubstituted alkyl, or substituted or unsubstituted phenyl],—NHC(═O)alkyl, —N[substituted or unsubstituted alkyl]C(═O)[substitutedor unsubstituted alkyl], —NHC(═O)[substituted or unsubstituted alkyl],—C(OH)[substituted or unsubstituted alkyl]₂, and —C(NH₂)[substituted orunsubstituted alkyl]₂. In another embodiment, by way of example, anoptional substituent is selected from oxo, fluorine, chlorine, bromine,iodine, —CN, —NH₂, —OH, —NH(CH₃), —N(CH₃)₂, —CH₃, —CH₂CH₃, —CH(CH₃)₂,—CF₃, —CH₂CF₃, —OCH₃, —OCH₂CH₃, —OCH(CH₃)₂, —OCF₃, —OCH₂CF₃,—S(═O)₂—CH₃, —C(═O)NH₂, —C(═O)—NHCH₃, —NHC(═O)NHCH₃, —C(═O)CH₃, and—C(═O)OH. In yet one embodiment, the substituents are independentlyselected from the group consisting of C₁₋₆ alkyl, —OH, C₁₋₆ alkoxy,halo, amino, acetamido, oxo and nitro. In yet another embodiment, thesubstituents are independently selected from the group consisting ofC₁₋₆ alkyl, C₁₋₆ alkoxy, halo, acetamido, and nitro. As used herein,where a substituent is an alkyl or alkoxy group, the carbon chain may bebranched, straight or cyclic, with straight being preferred.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible sub-ranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Description

The invention is partly based on the successful production of asemi-pure extract containing a compound of interest that has been testedin food applications including beverages, confectionery, dry mixes, bakegoods, and the like. Accordingly, the invention provides compositionsand methods of using a compound as a natural food colorant. In anotherembodiment, the compound of the invention can be used in cosmeticsettings. In one embodiment, the compound of the invention provides anadvantage to existing food colorants in the art. For example, thecompound of the invention is significantly more stable to heat, light,and oxygen, more vibrant, and less toxic.

Compounds

The compounds of the present invention may be synthesized usingtechniques well-known in the art of organic synthesis. The startingmaterials and intermediates required for the synthesis may be obtainedfrom commercial sources or synthesized according to methods known tothose skilled in the art.

Alternatively, the compounds of the present invention may be isolatedfrom avocado seed extract. Thus, the present invention provides a methodfor isolating compounds from avocado seed extract. In one embodiment,the method comprises blending avocado seeds, filtering the supernatant,lyophilizing the filtered supernatant, performing a first purificationusing flash chromatography, performing a second purification using anHPLC C18 column, eluting with a gradient of acetic acid andacetonitrile, performing a third purification using an HPLC Ultra Aromaxcolumn, eluting with a gradient of acetic acid and methanol, andobtaining an isolated compound

In one embodiment, the invention is a benzotropolone or a benzotropolonederivative. In one embodiment, the benzotropolone is substituted with asugar group. In one embodiment, the benzotropolone is substituted withan alkoxy-sugar group. In one embodiment, the benzotropolone issubstituted with a monosaccharide. In one embodiment, the benzotropoloneis substituted with a disaccharide. In one embodiment, thebenzotropolone is substituted with a trisaccharide.

In one embodiment, the invention is a compound of general formula (A):

wherein in general formula (A),

R¹ to R⁸ are each independently selected from the group consisting ofhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heterocyclyl, substituted heterocyclyl, heteroaryl,substituted heteroaryl, (C(R⁹R¹⁰))_(n), (C(R⁹R¹⁰))_(n)OR¹¹,(C(R⁹R¹⁰))_(n)(NR¹²)R¹¹, N(R⁹R¹⁰), and OR⁹, wherein any two of R¹ to R⁸are optionally joined to form a ring, wherein the ring is optionallysubstituted;

each occurrence R⁹ and R¹⁰ is independently selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, aryl, substituted aryl, heterocyclyl, substitutedheterocyclyl, heteroaryl, and substituted heteroaryl, wherein R⁹ and R¹⁰are optionally joined to form a ring, wherein the ring is optionallysubstituted;

each occurrence R¹² is independently selected from the group consistingof hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heterocyclyl, substituted heterocyclyl, heteroaryl,substituted heteroaryl, a monosaccharide, a disaccharide, and apolysaccharide;

each occurrence R¹² is independently selected from the group consistingof hydrogen alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heterocyclyl, substituted heterocyclyl, heteroaryl,and substituted heteroaryl;

each occurrence of n is independently an integer from 0 to 10; and

X is selected from the group consisting of O, NH and S.

In one embodiment, R³ and R⁵ are joined to form a ring, wherein the ringis optionally substituted. In one embodiment, the ring formed by R³ andR⁵ is a bicyclic ring. In one embodiment the ring is a five memberedring. In one embodiment, the ring is a six membered ring. In oneembodiment, the ring is a seven membered ring. In one embodiment, thering comprises a heteroatom. In one embodiment, the ring is ahydrocarbon ring.

In one embodiment R⁸ is hydroxyl.

In one embodiment X is O.

In one embodiment, R¹ is (C(R⁹R¹⁰))_(n)OR¹¹. In one embodiment, R¹ is(CH₂)(C)R⁹R¹⁰)(CH₂)₂OR¹¹. In one embodiment, R⁹ is C(═O)OH. In oneembodiment, R¹⁰ is C(═O)OH. In one embodiment R⁹ and R¹⁰ are joined toform a ring. In one embodiment, the ring comprises an O atom. In oneembodiment, the ring comprises one or more carbonyls. In one embodiment,the ring is a 3, 4 or 5 membered ring. In one embodiment R¹¹ is amonosaccharide. In one embodiment, R¹¹ is glucose, fructose orgalactose.

In one embodiment the compound of general formula (A) is a compound ofgeneral formula (B):

The compound of claim 1, wherein general formula (A) is represented bygeneral formula (B)

wherein, in general formula (B),

R² to R⁸ are each independently selected from the group consisting ofhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heterocyclyl, substituted heterocyclyl, heteroaryl,substituted heteroaryl, (C(R⁹R¹⁰))_(n), (C(R⁹R¹⁰))_(n)OR¹¹,(C(R⁹R¹⁰))_(n)(NR¹²)R¹¹, N(R⁹R¹⁰), and OR⁹, wherein any of R¹ to R⁸ areoptionally joined to form a ring, wherein the ring is optionallysubstituted;

each occurrence R⁹ and R¹⁰ is independently selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, aryl, substituted aryl, heterocyclyl, substitutedheterocyclyl, heteroaryl, and substituted heteroaryl, wherein R⁹ and R¹⁰are optionally joined to form a ring wherein the ring is optionallysubstituted;

each occurrence R¹¹ is independently selected from the group consistingof hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heterocyclyl, substituted heterocyclyl, heteroaryl,substituted heteroaryl, a monosaccharide, a disaccharide, and apolysaccharide;

each occurrence R¹² is independently selected from the group consistingof hydrogen alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heterocyclyl, substituted heterocyclyl, heteroaryl,and substituted heteroaryl;

each occurrence of n is independently an integer from 0 to 10;

m is an integer from 1 to 11;

p is an integer from 0 to 5; and

X is selected from the group consisting of O, NH and S.

In one embodiment, the compound of general formula (A) is a compound ofgeneral formula (C):

The compound of claim 1, wherein general formula (A) is represented bygeneral formula (C)

wherein in general formula (C),

R¹, R², R⁴, and R⁶-R⁸ are each independently selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, aryl, substituted aryl, heterocyclyl, substitutedheterocyclyl, heteroaryl, substituted heteroaryl, (C(R⁹R¹⁰))_(n),(C(R⁹R¹⁰))_(n)OR¹¹, (C(R⁹R¹⁰))_(n)(NR¹²)R¹¹, N(R⁹R¹⁰), and OR⁹, whereinany of R¹, R², R⁴, and R⁶-R⁸ are and OR⁹, wherein any of R¹, R², R⁴, andR⁶-R⁸ are optionally joined to form a ring, wherein the ring isoptionally substituted;

each occurrence R⁹ and R¹⁰ is independently selected from the groupconsisting of hydrogen, an alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heterocyclyl,substituted heterocyclyl, heteroaryl, and substituted heteroaryl,wherein R⁹ and R¹⁰ are optionally joined to form a ring;

each occurrence R¹¹ is independently selected from the group consistingof hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heterocyclyl, substituted heterocyclyl, heteroaryl,substituted heteroaryl, a monosaccharide, a disaccharide, and apolysaccharide;

each occurrence R¹² is independently selected from the group consistingof hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heterocyclyl, substituted heterocyclyl, heteroaryl,and substituted heteroaryl;

each occurrence of n is independently an integer from 0 to 10;

X is selected from the group consisting of O, NH and S; and

A is an optionally substituted 3 to 10 membered ring.

In one embodiment, the compound is

In one embodiment, the compound has a color. In one embodiment, thecompound is yellow, orange or red.

In one embodiment, the invention is a compound of general formula (I):

wherein in general formula (I),

R¹ is selected from the group consisting of CH₃, CH₂OH, —C(═O)OH,—C(═NH)OH, —C(═O)NH₂, —C(═NH)NH₂, —OH, and —NH₂;

R² is selected from the group consisting of H, CH₃, OH, —NH₂, —C(═O)OH;

R³ is selected from the group consisting of H, CH₃, OH, amonosaccharide, a disaccharide, and a polysaccharide;

A is a cycloalkyl ring having from 5 or 6 ring atoms, wherein thecycloalkyl ring may optionally have 0 to 3 double bonds;

each occurrence of R⁴ is independently selected from the groupconsisting of H, alkyl, substituted alkyl, alkenyl, aryl, substitutedaryl, and OH, wherein two adjacent R⁴ are optionally joined together toform a ring having 5 to 6 ring atoms, wherein the ring is optionallysubstituted;

L¹ and L² are each independently selected from the group consisting of asingle bond, alkyl, alkenyl, aryl, cycloalkyl, alkylaryl, andalkylcycloalkyl, wherein the alkyl, aryl, cycloalkyl, alkylaryl, oralkylcycloalkyl group is optionally substituted;

each occurrence of X is independently selected from the group consistingof O, NH, and S; and

n is an integer from 0 to 6.

In one embodiment, R¹ is C(═O)OH.

In one embodiment, L¹ is an alkyl. In other embodiments L¹ is analkenyl. In certain embodiments, L¹ is selected from the groupconsisting of —(CH₂)_(n2)— and —(CH═CH)_(n3)—. In certain embodiments L¹is CH₂. In another embodiment L¹ is CH═CH. In another embodiment L¹ is(CH═CH)₂. In yet another embodiment L¹ is (CH═CH)₃.

In one embodiment, R² is OH.

In some embodiments R³ is a monosaccharide. In one embodiment, themonosaccharide is glucose. In another embodiment, the monosaccharide isfructose. In yet another embodiment, the monosaccharide is galactose.

In one embodiment L² is CH₂. In another embodiment, L² is (CH₂)₂. In yetanother embodiment, L² is (CH₂)₃.

In one embodiment, X is O.

In one embodiment, A is a cycloalkyl ring having 6 ring atoms. In oneembodiment the cycloalkyl ring having 6 ring atoms has 1 double bond. Inanother embodiment, cycloalkyl ring having 6 ring atoms has 2 doublebonds. In yet another embodiment, cycloalkyl ring having 6 ring atomshas 3 double bonds.

In one embodiment, R⁴ is OH. In another embodiment, two adjacent R⁴ arejoined together to form a 5-membered ring, wherein one of the R⁴ is O.In certain embodiments, n is 2. In another embodiment, n is 3.

In certain embodiments, the compound of general formula (I) is acompound of general formula (II):

wherein in general formula (II),

R¹ is selected from the group consisting of CH₃, CH₂OH, —C(═O)OH,—C(═NH)OH, —C(═O)NH₂, —C(═NH)NH₂, —OH, and —NH₂;

R² is selected from the group consisting of H, CH₃, OH, —NH₂, —C(═O)OH;

R³ is selected from the group consisting of H, CH₃, OH, amonosaccharide, a disaccharide, and a polysaccharide;

each occurrence of R^(4a), R^(4b), R^(4c), and R⁵ is independentlyselected from the group consisting of H, alkyl, substituted alkyl, aryl,substituted aryl, and OH;

L¹ is selected from the group consisting of —(CH₂)_(n2)— and—(CH═CH)_(n3)—;

X is selected from the group consisting of O, NH and S;

n¹ is an integer from 0 to 6;

n² is an integer from 0 to 6, and

n³ is an integer from 0 to 3.

In some embodiments, R^(4b) is OH. In another embodiment, R^(4a) is H.In yet another embodiment, R^(4c) is H.

In some embodiments n¹ is 2. In other embodiments n¹ is 3.

In one embodiment, R⁵ is H.

In some embodiments, the compound of general formula (II) is selectedfrom the group consisting of

In certain embodiments, the compound of general formula (I) is acompound of general formula (III):

wherein,

R¹ is selected from the group consisting of CH₃, CH₂OH, —C(═O)OH,—C(═NH)OH, —C(═O)NH₂, —C(═NH)NH₂, —OH, and —NH₂;

R² is selected from the group consisting of H, CH₃, OH, —NH₂, —C(═O)OH;

R³ is selected from the group consisting of H, CH₃, OH, amonosaccharide, a disaccharide and a polysaccharide;

each occurrence of R⁴ is independently selected from the groupconsisting of H, alkyl, substituted alkyl, alkenyl, aryl, substitutedaryl, and OH, wherein two adjacent R⁴ are optionally joined together toform a ring having 5 to 6 ring atoms, wherein the ring is optionallysubstituted;

each occurrence of X is independently selected from the group consistingof O, NH, and S;

n¹ is an integer from 0 to 6; and

n² is an integer from 0 to 6.

In one embodiment R⁴ is OH. In another embodiment R⁴ is selected fromthe group consisting of (CH═CH)OH, (CH═CH)₂OH and (CH═CH)₃OH.

In some embodiments, two adjacent R⁴ are joined together to form a ringhaving 5 ring atoms, wherein a first R⁴ is O and a second R⁴ is C.

In some embodiments, the compound of general formula (III) is selectedfrom the group consisting of

In another embodiment, the compound of general formula (I) is

In one aspect, the invention provides a compound of general formula(IV):

wherein general formula (IV),

Q is selected from the group consisting of (CH₂)_(m3), (CH═CH)_(m4), and(CH₂)_(m5)(C═O)O(C═O)(CH₂)_(m6);

R^(2′) is selected from the group consisting of H, CH₃, OH, —NH₂, and—C(═O)OH;

R^(3′) is selected from the group consisting of H, CH₃, OH, amonosaccharide, a disaccharide, and a polysaccharide;

C and D are each independently a cycloalkyl ring having from 5 or 6 ringatoms, wherein the cycloalkyl ring may optionally have 0 to 3 doublebonds;

each occurrence of R^(4′) is independently selected from the groupconsisting of H, alkyl, substituted alkyl, aryl, substituted aryl, andOH, wherein two adjacent R⁴ are optionally joined together to form aring having 5 to 6 ring atoms, wherein the ring is optionallysubstituted;

each of L^(1′) and L^(2′) is selected from the group consisting of asingle bond, alkyl, aryl, cycloalkyl, alkylaryl, and alkylcycloalkyl,wherein the alkyl, aryl, cycloalkyl, alkylaryl, or alkylcycloalkyl groupis optionally substituted;

each occurrence of X is independently selected from the group consistingof O, NH and S;

R^(7′) is selected from the group consisting of H, CH₃, OH, —NH₂, and—C(═O)OH;

R^(8′) is selected from the group consisting of H, CH₃, OH, amonosaccharide, a disaccharide, and a polysaccharide;

each occurrence of R^(9′) is independently selected from the groupconsisting of H, alkyl, substituted alkyl, aryl, substituted aryl, andOH, wherein two adjacent R⁴ are optionally joined together to form aring having 5 to 6 ring atoms, wherein the ring is optionallysubstituted;

m₁ is an integer from 0 to 6;

m₂ is an integer from 0 to 6

m₃ is an integer from 0 to 3

m₄ is an integer from 0 to 3;

m₅ is an integer from 0 to 3; and

m₆ is an integer from 0 to 3.

-   -   In one embodiment R^(2′)

In one embodiment, R^(7′) is OH.

In certain embodiments, R^(3′) is a monosaccharide. In one embodiment,the monosaccharide is glucose. In another embodiment, the monosaccharideis fructose. In yet another embodiment, the monosaccharide is galactose.

In certain embodiments, R^(8′) is a monosaccharide. In one embodiment,the monosaccharide is glucose. In another embodiment, the monosaccharideis fructose. In yet another embodiment, the monosaccharide is galactose.

In one embodiment, C is a cycloalkyl ring having 6 ring atoms. In oneembodiment the cycloalkyl ring having 6 ring atoms has 1 double bond. Inanother embodiment, cycloalkyl ring having 6 ring atoms has 2 doublebonds. In yet another embodiment, cycloalkyl ring having 6 ring atomshas 3 double bonds.

In one embodiment, D is a cycloalkyl ring having 6 ring atoms. In oneembodiment the cycloalkyl ring having 6 ring atoms has 1 double bond. Inanother embodiment, cycloalkyl ring having 6 ring atoms has 2 doublebonds. In yet another embodiment, cycloalkyl ring having 6 ring atomshas 3 double bonds.

In one embodiment, R^(4′) is OH. In another embodiment, two adjacentR^(4′) are joined together to form a 5-membered ring. In one embodiment,two adjacent R^(4′) are joined together to form a 5-membered ring,wherein at least one R^(4′) is O.

In one embodiment, R^(9′) is OH. In another embodiment, two adjacentR^(9′) are joined together to form a 5-membered ring. In one embodiment,two adjacent R^(4′) are joined together to form a 5-membered ring,wherein at least one R^(9′) is O.

In one embodiment L^(1′) is CH₂. In another embodiment, L^(1′) is(CH₂)₂. In yet another embodiment, L^(1′) is (CH₂)₃.

In one embodiment L^(2′) is CH₂. In another embodiment, L^(2′) is(CH₂)₂. In yet another embodiment, L^(2′) is (CH₂)₃.

In one embodiment Q is R¹⁰ (C═O)O(C═O)R¹¹.

In some embodiments R¹⁰ is selected from an alkyl, alkenyl, aryl,cycloalkyl, alkylaryl, and alkylcycloalkyl, wherein the alkyl, aryl,cycloalkyl, alkylaryl, or alkylcycloalkyl group is optionallysubstituted. In one embodiment R¹⁰ is an alkyl. In yet anotherembodiment R¹⁰ is CH₂.

In some embodiments R¹¹ is selected from an alkyl, alkenyl, aryl,cycloalkyl, alkylaryl, and alkylcycloalkyl, wherein the alkyl, aryl,cycloalkyl, alkylaryl, or alkylcycloalkyl group is optionallysubstituted. In one embodiment R¹¹ is an alkyl. In yet anotherembodiment R¹¹ is CH₂.

In one embodiment Q is CH₂(C═O)O(C═O)CH₂.

In one embodiment, the compound of general formula (IV) is

Compound Preparation

In one embodiment, the invention provides compound prepared by a processcomprising the following steps: A compound prepared by a processcomprising the steps of:

obtaining a seed of Persea americana; grinding size reduction of theseed to obtain a slurry; incubating the slurry; extracting the compoundby incubating the slurry with an alcohol to form a first mixture;isolating a first substance from the first mixture; removing theinsoluble particles from the first substance; precipitating thesubstance to form a second mixture; isolating a second substance fromthe second mixture; adsorbing the second substance to a resin; andisolating the compound by eluting the compound from the resin with analcohol.

In one embodiment, the alcohol is methanol, ethanol, acetone, citricacid, acetic acid or any combination thereof. In one embodiment, thealcohol is diluted in water.

In one embodiment, the step grinding size reduction of the seedcomprises two steps, a course size reduction step and a second finereduction step.

In one embodiment, the step incubating the slurry comprises incubatingthe slurry for at least one minute. In one embodiment, the incubation isfor more than 30 minutes. In one embodiment, the incubation is up to 48hours. In one embodiment, the step incubating the slurry comprisesincubating the slurry for at 0-40° C. In one embodiment, the incubationis at 20-40° C. In one embodiment, the incubation is at 20° C.

In one embodiment, the step isolating a first liquid from the firstmixture comprises centrifugation or filtration through a filter.

In one embodiment, the step removing the insoluble particles from thefirst substance comprises filtration through a filter.

In one embodiment, precipitating the slurry comprises incubating theslurry for at least 24 hours and up to 48 hours. In one embodiment,incubating the substance comprises incubating the liquid at 4° C.

In one embodiment, the step isolating a second substance from the secondmixture comprises filtration or centrifugation.

In one embodiment, the step adsorbing the second substance to a resincomprises applying the liquid to a XAD-7 resin. In one embodiment, theresin is washed twice.

In one embodiment, the compound is isolated by eluting the compound fromthe resin with an alcohol. In one embodiment compound is concentrated byevaporation. In one embodiment the compound is dried by freeze drying orspray drying. In one embodiment, the dried compound is mixed with anexcipient. In one embodiment the excipient is maltodextrin or sugar.

Salts of the Compounds of the Invention

The compounds described herein may form salts with acids or bases, andsuch salts are included in the present invention. The term “salts”embraces addition salts of free acids or free bases that are compoundsof the invention.

Compositions of the Invention

The invention includes an edible composition comprising a compound ofthe invention. In one embodiment, the compound of the invention in theedible material is present in an amount from about 0.25 mg/mL to about10 mg/mL. In one embodiment, the edible material comprising a compoundof the invention has a hue selected from the group consisting of red,orange and yellow.

In one aspect of the invention, compounds of the invention may becombined with one or more natural or artificial food colorants such asthose approved by the U.S. Food and Drug Administration(http://www.fda.gov/ForIndustry/ColorAdditives/ColorAdditiveInventories/ucm115641.htm).In one embodiment, the natural food colorant includes, but is notlimited to Citrus Red #2, safranol curcumin, capsaicin, β-carotene,bixin, and carmine, annato extract, dehydrated beets, canthaxanthin,caramel, β-apo-8′-carotenal, cochineal extract, carmine, sodium copperchlorophyllin, toasted partially defatted cooked cottonseed flour,ferrous gluconate, ferrous lactate, grape color extract, synthetic ironoxide, fruit juice, vegetable juice, carrot oil, paprika, paprikaoleoresin, mica-based pearlescent pigments, riboflavin, saffron,spirulina extract, titanium dioxide, tomato lycopene extract, tomatolycopene concentrate, turmeric, and turmeric oleoresin.

In another embodiment, the artificial food colorant includes but is notlimited to FD&C Blue #1, FD&C Blue #1 Aluminum Lake, FD&C Blue #2, FD&CBlue #2 Aluminum Lake on alumina, FD&C Green #3, FD&C Red #3, FD&C Red#40 and its Aluminum Lake, FD&C Yellow #5, FD&C Yellow #5 Aluminum Lake,FD&C Yellow #6, FD&C Yellow #6, FD&C Yellow #6 Aluminum Lake, titaniumcomplexes, and Orange B.

In one aspect, the composition of the invention further comprises analuminum-containing compound, to form an aluminum lake, wherein theunpleasantness of the taste and/or odor of the coloring material isreduced by said combination with the aluminum-containing compound. Inanother aspect, the composition of the invention further comprisescalcium.

In another embodiment, the composition of the invention furthercomprises a diluent and is in a form including, but not limited to,liquids, powders, gels, and pastes.

In one aspect, the composition of the invention could be an extract ofavocado seeds. In another aspect, the composition is freeze-dried orspray-dried.

Methods of the Invention

In one aspect, the present invention provides methods for coloring amaterial. In one embodiment, the material is an edible material, a foodproduct, a cosmetic product, a drug product or a medical device. Incertain embodiments, the material is orange. In other embodiments, thematerial is yellow. In yet another embodiment, the material is red. Inone embodiment, the method for coloring a material comprises adding acompound of the invention to the material.

In one embodiment, the method further comprises adding a compound of theinvention to the edible material at a desired concentration. In oneembodiment, the concentration is from about 0.25 mg/mL to about 10mg/mL. In one embodiment, the concentration is from about 1 ppm to 10ppm. In one embodiment the concentration is from about 1 ppm to 100 ppm.In another embodiment the concentration is from about 1 ppm to 1000 ppm.In yet embodiment the concentration is from about 1 ppb to 10 ppb. Inyet embodiment the concentration is from about 1 ppb to 100 ppb. In yetembodiment the concentration is from about 1 ppb to 500 ppb.

In some embodiments, the invention provides a method of imparting acolor to a substrate. In some embodiments, the method of imparting ared, orange or yellow color to a substrate (e.g., a food item, acosmetic, a drug or nutraceutical product, a textile product, a devicesuch as a medical device) comprises contacting the substrate with acolorant composition comprising at least one compound of the inventiondescribed herein. In some embodiments, the colorant composition isprepared by mixing a compound herein with a color additive (e.g. a FDAapproved color additive). In some embodiments, the substrate is anedible material. In some embodiments, the substrate is a food item. Insome embodiments, the substrate is a medical device. In someembodiments, the substrate is a drug product. In some embodiments, thesubstrate is a nutraceutical product. In some embodiments, the substrateis a cosmetic product.

In certain embodiments, the amount of a colorant composition to beincorporated into a material depends on the final color to be achieved.In some embodiments, the food product, the cosmetic product, the drugproduct, the medical device, comprises a colorant composition disclosedherein in an effective amount, by itself or with another colorant, toimpart the edible material, food product, cosmetic product, drug productor medical device a color including, but not limited to orange, yellowand red.

In one embodiment, the invention provides a method of coloring amaterial, wherein the color is a yellow hue, a red hue or an orange hue.

In one embodiment, the invention provides a method of coloring amaterial, wherein the color is a yellow hue, including, but not limitedto Amber, Apricot, Arylide yellow, Aureolin, Beige, Buff, Cadmiumpigments, Chartreuse, Chrome yellow, Citrine, Citron, Color term, Cream,Dark goldenrod, Diarylide pigment, Ecru, Flax, Fulvous, Gamboge, Gold,Goldenrod, Hari, Harvest gold, Icterine, Isabelline, Jasmine, Jonquil,Khaki, Lemon, Lemon chiffon, Lime, Lion, Maize, Marigold, Mikado yellow,Mustard, Naples yellow, Navajo white, Old gold, Olive, Or (heraldry),Peach, Pigment Yellow 10, Pigment Yellow 16, Pigment Yellow 81, Pigmentyellow 83, Pigment yellow 139, Saffron, Sage, School bus yellow,Selective yellow, Stil de grain yellow, Straw, Titanium yellow,Urobilin, or Vanilla.

In one embodiment, the invention provides a method of coloring amaterial, wherein the color is a red hue, including, but not limited to,Scarlet, Imperial red, Indian red, Spanish red, Desire, Lust, Carmine,Ruby, Crimson, Rusty red, Fire engine red, Cardinal red, Chili red,Cornell Red, Fire brick, Redwood, OU Crimson, Dark red, Maroon, Barnred, and Turkey red.

In one embodiment, the invention provides a method of coloring amaterial, wherein the color is an orange hue, including, but not limitedto, Papaya whip, Peach, Apricot, Melon, Atomic tangerine, Tea rose,Carrot orange, Orange peel, Princeton orange, UT Orange, Spanish orange,Tangerine, Pumpkin, Giants orange, Vermilion (Cinnabar), Tomato,Bittersweet, Persimmon, Persian orange, Alloy orange, Burnt orange,Bittersweet shimmer, Brown. In one embodiment the yellow hue has awavelength from 585 nm-620 nm.

The effectiveness of the colorant composition can be determined bycomparing (e.g., by visual comparison) a color to be achieved (e.g., ared) with the product or device colored with an amount of the colorantcomposition.

In one aspect, the compounds of the invention can be used in cosmeticsettings. In another aspect of the invention the compounds can be usedfor coloring drugs. In yet another application, the compounds can beused to color nutritional supplements.

EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations that are evident as a result of the teachings providedherein.

Example 1 Characterization of a Natural Orange Pigment Found in HassAvocado (Persea americana) Seed for Use as a Natural Food Colorant

Avocado seed extract represents a novel source of yellow-orange-rednatural colors, which are stable in a variety of conditions. The use ofcolored avocado seed extracts are particularly appealing for productswith long shelf-lives such as beverages and candies, as well as productswhich are baked or undergo pasteurization. Using avocado seed extract asa natural colorant will provide a new value-added use for avocado seedswhich are typically viewed as a low-value waste product.

There is a great demand presently for natural colors to replaceartificial colorants. However, a large limitation for natural colorantsis their instability. The data presented herein provides naturalcompounds and derivatives thereof which have greater color intensity andare significantly more stable to heat, light and oxygen than othernatural colorants, especially those in the yellow-red range. Moreover,because of the superior stability, the compounds of the presentinvention are less toxic.

The materials and methods employed in the experiments presented in thisExample are now described.

Preparation of a Semi-Pure Colored Avocado Seed Extract

Hass avocados, Great Value Pure Cane Sugar, Sprite, Great Value 100%Apple Juice, Ocean Spray 100% White Grapefruit Juice, PillsburyTradition Vanilla Cake Mix, and other baking food materials werepurchased from local grocery stores. Chemicals used were reagent gradeand were used as supplied except where noted. Lyophilization wasperformed using a Virtis Genesis 25 XL Pilot Lyophilizer (Warminster,PA). Organic solvents were removed using a rotary evaporator (Heidolph;Germany). L*a*b* values were determined using a Minolta CR- 200 ChromaMeter (Japan). All LC samples were filtered using 25 mm syringe filterwith 0.45 μm cellulose acetate membrane (VWR; Radnor, Pa.). All otherreagents were of the highest quality available.

After removal from the avocados, seeds were cleaned, peeled, and choppedby hand into small pieces. The pieces were then were blended withdeionized, distilled water in a laboratory blender (Waring; Wilmington,N.C.) for 60 s. The resulting seed/water mixture was placed in therefrigerator at 4° C. for 24 h. After 24 h, the supernatant was gravityfiltered through blotting paper (grade 703, VWR). The filteredsupernatant was frozen in plastic trays and lyophilized to produce adried, raw extract (˜3.73% yield). The dried raw extract was furtherpurified by flash chromatography using a nitrogen pressurized (<2ppmmoisture, Penn State General Stores) glass column packed with amberliteXAD7-HP (Sigma; Cleveland, Ohio). The column was eluted with water toremoved sugars from the extract, and a colored fraction eluted usingmethanol containing 0.1% (v/v) acetic acid. The organic solvent wasremoved using a rotary evaporator, and the remaining water frozen andlyophilized to produce a semi-pure colored extract (˜29% yield). Thissemi-pure extract was used in all color and stability studies.

Color and Stability of Semi-Pure CASE in Some Commercial and Model FoodProducts

Color Studies

Semi-pure CASE (colored avocado seed extract) was added to Sprite (pH3.29), apple juice (pH 3.71), white grapefruit juice (pH 3.25), andwhite cupcake mix at final concentrations of 0, 0.25, 0.75, 1, 3, 5, 8,and 10 mg/mL. For the cupcakes, L*a*b* values were determined afterbaking and were measured on both the middles and tops of the cakes. Foreach food product, L*a*b* values were found and ΔE values calculatedusing the uncolored food product as the control and using the equation

ΔE=√{square root over ((L ₀ −L)²+(a₀ −a)²+(b ₀ −b)²)}

where L₀, a₀, and b₀ are the respective values of the uncolored foodsample.

To prepare cheese sauces, regular orange and color-additive free whiteKraft cheese powders were provided by Wincrest Bulk Foods (Munnsville,N.Y.). Orange cheese powders were prepared using 1, 5, 10, and 20% byweight semi-pure CASE in the white cheese powder. L*a*b* values weredetermined for each dry cheese powder as well as prepared cheese sauce(1 g cheese powder combined with 2 mL warm, skim milk). L*a*b* valueswere used to calculate ΔE for each sample, with white cheese powder usedas the control

Stability Studies in Model Sugar Drinks

Samples were prepared by adding semi-pure CASE to a model sugar drinksolution (2.6 M sodium citrate buffer containing 500 g/L sucrose). Thesolutions were adjusted to either pH 2.5 or pH 5.85. Semi-pure CASE wasadded to final concentrations of 0, 1, and 5 mg/mL. The samples wereprepared in duplicate and placed into screw-cap GC vials. After beingsealed into the GC vials, samples were bubbled with nitrogen to removeoxygen from the sample and the headspace. The samples were then dividedinto four treatment groups and sampled as outlined in Table 1. Sampletesting at each time point included pH determination, L*a*b* values, andUV-Vis spectroscopy. At each time point, a 3 mL aliquot was removed fromeach sample using a gas-tight syringe while bubbling nitrogen throughthe sample in order to retain an oxygen free environment.

TABLE 1 Sampling time points (days) on which each treatment group wassampled. Dark, Dark, Dark, Light, 40° C. 23° C. 4° C. 26° C. 1  1 —  1 2 2 —  2 3 — — — 4 — — — 5 — — — 6 — — — 7 — — — 8  8  8  8 — 15 15 15 —21 21 21 — 29 29 29 — 37 37 37 — 43 43 43 — 50 50 — — 56 56 —

Effects of pH on the Color of CASE

Two identical samples of semi-pure colored extract were prepared bydissolving 0.05 g of the extract in 10 mL distilled, deionized water.Samples were stirred with stir plates. The native pH of the treatmentand control samples was 3.32 and 3.42, respectively. The treatmentsample was treated with 10 M NaOH until the sample reached pH 12.32. Thetreatment sample was then treated with 6 M HCl until pH 1.59 wasreached. In all cases an equivalent volume of water was added to thecontrol sample in order to maintain similar concentrations. The final pHof the control sample was 3.50, and the treatment sample was finallyadjusted to pH 3.57.

Preparation of Colored and Uncolored Avocado Seed Extracts forMetabolomics

Five biological replicates were prepared of both colored and uncoloredextracts. Each replicate contained approximate 10 g portions from twoavocado seeds, totaling 20 g of seed per replicate. Colored replicateswere prepared by blending -20 g of seeds into 400 mL of deionized,distilled water. For uncolored replicates, ˜20 g of seeds was blendedinto 400 mL of deionized distilled water containing tropolone (5.0 mg,0.041 mmol). Studies were completed at the Penn State Metabolomics Corefacility with the help of Phillip Smith, in the laboratory of AndrewPatterson. LC-MS/MS was completed using a Shimadzu (Kyoto, Japan)Prominence UFLC and an AB SCIEX (Framingham, Mass.) 5600 Triple TOF MassSpectrometer and principal component analysis (PCA) was performed usedSIMCA P statistical package.

Structure Elucidation of the Colored Compound

HPLC Purification

The semi-pure, post-amberlite CASE was further purified using an AgilentPrepStar system with 440-LC fraction collector (Santa Clara, Calif.).The extract was dissolved in deionized, distilled water to a finalconcentration of 20 mg/mL and filtered. Samples (10 mL) were injectedonto a Viva C18 250 mm×10 mm×5 μm column (Restek, Bellefonte, Pa.).Samples were separated using a gradient of deionized water containing0.1% acetic acid and acetonitrile. The percentage of acetonitrileincreased with time as follows; 0 min, 5%; 0-40 min, 5-30%; 40-45 min,30-95%; 45-48 min, 95%; 48-49 min, 95-5%; 49-51 min, 5% at a flow rateof 4 mL/min. Fractions were collected at 30 s intervals (2 mL each) from19.5 min to 26 min. The peak of interest, F12, eluted at approximately22 min. All subsequent fractions containing F12 were combined andlyophilized to produce “rough F12.” In some instances, F12 as referredelsewhere herein, may have an IUPAC names as follows:2-(4-hydroxy-8-(2-((5-hydroxy-2-oxo-2,6,7,7a-tetrahydrobenzofuran-6-yl)oxy)ethyl)-5-oxo-6-(((2R,4R,5R)-3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-yl)methoxy)-5H-benzo[7]annulen-3-yl)aceticacid.

Once dried, the rough F12 samples were diluted with deionized water and10 mL samples were injected onto an Ultra Aromax 250 mm×10 mm×5 μmcolumn (Restek). Samples were resolved using a gradient method ofdeionized water containing 0.1% acetic acid, and methanol. Thepercentage of methanol was increased as a function of time as follows: 0min, 48%; 0-13.5 min, 48-65%; 13.5-14.5 min, 65%; 14.5-15 min, 65-48%;15-17 min, 48%, at a flow rate of 4 mL/min. Fractions were collected at24 sec intervals (1.6 mL each from 8.9 min to 14.5 min. The peak ofinterest eluted as the later of 2 overlapping peaks at approximately10.6 min to produce pure F12.

Pure F12 fractions were combined and lyophilized. 20 μL injections weremade onto an ultra Aromax 150 mm×4.6 mm×5μm column (Restek; Bellefonte,Pa.). A gradient method was used with solvent being filtered DDI waterwith 0.1% acetic acid and solvent B being methanol. The method was asfollows: 0 min, 45%; 0-25, 45-62%; 25-28 min, 62%; 28-29 min, 62-45%;29-31 min, 45%. If additional purification was necessary, the fractionswere again fractionated using the PrepStar system.

High Resolution MS/MS Analysis

Samples (5u1) were separated by reverse phase HPLC using a Prominence 20UFLCXR system (Shimadzu, Columbia Md.) with a Waters (Milford, Mass.)BEH C18 column (100 mm×2.1 mm×1.7 um particle size) maintained at 55° C.and a 20 minute aqueous acetonitrile gradient, at a flow rate of 250ul/min. Solvent A was HPLC grade water with 0.1% formic acid and SolventB was HPLC grade acetonitrile with 0.1% formic acid. The initialcondition were 97% A and 3% B, increasing to 45% B at 10 min, 75% B at12 min where it was held at 75% B until 17.5 min before returning to theinitial conditions. The eluate was delivered into a 5600 (QTOF)TripleTOF using a Duospray™ ion source (all AB Sciex, Framingham,Mass.). The capillary voltage was set at 5.5 kV in positive ion mode and4.5 kV in negative ion mode, with a declustering potential of 80V. Themass spectrometer was operated in IDA (Information DependentAcquisition) mode with a 100 ms survey scan from 100 to 1200 m/z, and upto 20 MS/MS product ion scans (100 ms) per duty cycle using a collisionenergy of 50V with a 20V spread. Principal component analysis wasprocessed using square root mean square analysis. Known compounds wereidentified using the Scripps METLIN metabolomics database.

Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy(ATR)

ATR experiments were conducted using a Bruker (Billerica, Mass.) Vertex70v scanning the mid-IR range.

High Resolution NMR Analysis

Experiments used a Bruker Avance II HD 500 MHz NMR with LN₂ cryoprobe.1D ¹H and ¹³C experiments, as well as 2D Correlation Spectroscopy(COSY), Total Correlation Spectroscopy (TOCSY), Heteronuclear MultipleBond Correlation (HMBC), and Distortionless Enhancement by PolarizationTransfer (DEPT) edited Homonuclear Single bond Quantum Correlation(HSQC) experiments were conducted on two separate samples, one in D₂Oand one in (CD₃)₂SO. Additionally a 2D Nuclear Overhauser EffectSpectroscopy (NOESY) and selective TOCSY and Rotating Frame NOESY(ROESY) experiments were conducted on the D₂O sample. Data was processedusing Topspin and MestReNova software.

The results of the experiments presented in this Example are nowdescribed.

Colorant properties of semi-pure CASE in a panel of commercial foodproducts

Semi-pure CASE was added to three commercial beverages to assess thebehavior of the pigment in a variety of matrices (FIG. 1). Visually, thecolor of the extract appeared to be the most vibrant in the Sprite. FIG.2 shows the calculated ΔE values for the samples. When used in baking,the semi-pure CASE retained its colorant properties well, although itshould be noted that higher concentrations of the extract produced adenser crumb (FIG. 3A and FIG. 3B). Baked samples were prepared induplicate. The addition of semi-pure CASE showed a steady climb in ΔEvalues in both the tops and middles of the cupcakes (FIG. 4).

Semi-pure CASE was also added to white cheese powder (blank) andcompared to regular orange Kraft cheese powder (FIG. 5A). To create acheese sauce, warm milk (2 mL/g) was added to the cheese powders (FIG.5B). The CASE produced a strong orange color, but was not as bold orbright as the regular Kraft cheese powder. The ΔE values appear to reacha maximum around 100 mg/g dry sample or 33.3 mg/g prepared sample. Asimilar result was observed in the white cake mix around 8 mg/mLsemi-pure CASE.

Stability of Semi-Pure CASE in a Model Sugar Drink

Semi-pure CASE samples were prepared in a model sugar drink withconcentrations of 1 mg/mL or 5 mg/mL, and at pH 2.5 and pH 5.85.Treatment groups consisted of three dark groups at 4° C., 23° C., and40° C., and one treatment group of lighted samples at 26° C. Sampleswere prepared in duplicate, and L*a*b* measurements of each sample wereperformed twice. ΔE values for the samples can be seen in FIG. 7Athrough FIG. 7D. In general, the greater the ΔE value, the more likelyit is that the corresponding color change is perceptible to the humaneye. It is widely accepted that a change of ΔE≤2.5 is insignificant, orlikely to be imperceptible to the human eye (Salameh et al., 2014, Int JEsthet Dent 9:1-9). After deoxygenation by bubbling nitrogen throughsamples, a change in pH in the order of −0.35 pH units was observed forall samples. The pH of all samples then remained constant throughout theremainder of the experiment. Some variation in ΔE is expected due to thenature of the experiment. Apart from some minor fluctuations, samplesretained their bright colors even after 50 d. Photos of the samples onday 36 are shown below in FIG. 8. Samples were measured on thespectrometer during the experiment to determine any change in sampleabsorbance at 445 nm, the wavelength corresponding to the most prominentcolored compound (FIG. 9A through FIG. 9D). Though the light treatedsamples experienced a minor decrease in ΔE values over time, they showedno significant change in absorbance at 445 nm. Both the 1 mg/mL at pH5.85 and the 5 mg/mL samples at pH 2.5 samples experienced increases inΔE and absorbance at 445 in the darkened, room temperature samplesduring the final three weeks. This may have been due to formation of aprecipitate or improper sampling technique, as no such trend is observedin any of the other samples.

Effect of pH on Color of Semi-Pure CASE

The effect of pH on raw CASE samples has been previously reported(Dabas, et al., 2011. J. Food Sci 76:C1335-41; Dabas, 2012, Ph.D.Thesis, The Pennsylvania State University). Here, the effect of pH insemi-pure CASE is reported. Semi-pure CASE in water at a finalconcentration of 5 mg/mL has a pH of 3.32 and a yellow color (FIG. 10through FIG. 10C). Indeed, adjusting the pH to neutral levels againproduced a deeper orange color, and increasing the pH to 10-12 created adark red, and finally brownish red color. Upon returning the sample toits native pH range, and even lower to pH 1.59 produced a sample inwhich color was still pH dependent, but the color range had been shiftedto a more orange-red range. Spec data showed an increase in sampleabsorbance at both 445 nm and 480 nm (FIG. 11). LC profiles showed thatthe most prominent colored peak, F12, was still present after treatmentwith base. The 280 nm profile remained as well, though decreased, whilea substantial increase was seen in the 320 nm profile (FIG. 11 and FIG.12). This red-shift in color and increase in the 320 nm peaks indicatesthat there may be some extended conjugation forming in compounds whereconjugation may have previously been limited.

Principal Component Analysis (PCA) of Colored and Uncolored Avocado SeedExtracts

An uncolored avocado seed extract was prepared by inhibiting the actionof PPO through the addition of tropolone. By comparing biologicalreplicates of colored and uncolored extracts, it was possible todetermine masses unique to each sample. FIG. 13 shows the clustering ofmasses in samples analyzed in positive mode. Variation between samplesis common when analyzing natural products such as avocados, and thatvariation can be observed in this data by the divergence betweenclustering of replicates, as seen in FIG. 13.

Masses near the upper left tended to be present at higher concentrationsin the colored samples, while samples towards the lower right tended tobe present at higher concentrations in the uncolored samples. FIG. 14shows each unique mass found in the samples in positive mode. Theclustering of samples analyzed in negative mode is shown in FIG. 15,while the total principal component analysis is shown in FIG. 16. Againmasses near the upper left tended to be present at higher concentrationsin the colored samples, while samples towards the lower right tended tobe present at higher concentrations in the uncolored samples. Principalcomponent analysis showed approximately forty-nine masses unique to thecolored or uncolored extract. Abscisic acid, and perseitol, the 7-membersugar alcohol, were present in both extracts while epicatechin,catechin, procyanidin B2 and salidroside were found only in theuncolored extract. Table 2 shows a list of masses found to be unique toone the extracts.

TABLE 2 Compounds found in colored & uncolored avocado seed extracts viaprincipal component analysis. Extract Retention time Molecular(Compound) (min) Mode Ion Fragments Both 0.96 negative 211.082 193.0171,149.0457, (perseitol) 131.0353, 119.0347, 113.0243, 101.025, 89.0255,85.0309, 71.0163, 59.0173, 57.038, 55.0227 Both 4.05 positive 265.1413247.1325, 135.134, (abcsisic acid) 229.1225, 219.1386, 217.1219,211.1116, 203.1054, 196.0858, 187.1131, 175.0743, 161.0945, 147.0797,135.0791, 128.0619, 115.0552, 95.0498, 91.0547 Uncolored 4.51 negative289.073 271.0623, 247.0636, (epicatechin/ 245.0829, 227.0725, 221.0833,catechin) 205.0518, 203.0726, 187.0408, 161.0616, 159.0459, 151.0404,137.0252, 125.0247, 123.0456, 109.0303, 97.0303, 95.051 Uncolored 3.76negative 289.0726 245.0828, 123.0459, 109.031 (catechin/ epicatechin)Uncolored 4.17 positive 579.1484 439.1004, 427.1019, (Procyanidin B2)411.1085, 409.0885, 303.0826, 301.0698, 291.0857, 289.0682, 287.0547,259.0612, 247.0601, 229.0499, 215.0698, 205.0465, 201.0542, 191.0333,187.0373, 179.0321, 177.0547, 175.0398, 167.0334, 165.0542, 163.0382,159.0445, 149.0222, 147.0443, 139.0381, 135.0439, 127.039, 123.0435,109.0304, 68.9977 Uncolored 3.67 positive 323.1096 None (salidroside)uncolored 3.64 negative 299.1134 179.0547, 137.061, 119.0494, 101.0245,89.025, 71.0155 uncolored 3.64 negative 345.1193 299.1134, 179.0561,161.0457, 137.0613, 119.0439, 113.0249, 89.0255, 71.0157, 59.0168uncolored 2.45 negative 575.127 557.118, 531.1353, 513.41, 487.143,449.0897, 423.0757, 407.0825, 363.0927, 351.0499, 327.0516, 325.0733,309.0438, 307.0617, 287.0576, 243.0306, 241.0524, 217.0513, 175.041,167.0355, 125.0245 uncolored 3.86 negative 431.1571 299.1094, 191.0582,149.047, 119.05, 99.0113, 89.0259, 71.0144, 59.0145 uncolored 4.52negative 357.0588 311.0537, 289.0721, 245.0821, 203.0711, 137.0245,109.0302 uncolored 5.8 negative 437.0509 419.0418, 391.049, 285.3968,285.0378, 284.0348, 283.0264, 227.0345, 171.0448, 151.0035, 123.0059uncolored 4.16 negative 577.1356 451.1055, 425.0901, 407.0788, 339.0898,289.0725, 287.0565, 245.0819, 203.0691, 137.0238, 125.0244 uncolored3.43 negative 577.1423 559.1265, 457.1053, 425.0921, 407.0798, 339.0899,289.0736, 245.0829, 161.0252, 125.0248 uncolored 2.42 negative 863.1943711.1417, 693.1323, 649.1332, 575.1234, 513.123, 449.0925, 407.0818,297.0422, 287.0565, 243.0302, 167.0353 uncolored 6.04 negative 597.1882477.1443, 357.1041 345.1067, 339.0859, 315.0899, 233.0458, 209.0467,191.0366, 167.0354, 125.0244 uncolored 7.37 negative 540.149 494.1429,472.1618, 472.1854, 350.0873, 321.0949, 254.043, 232.0646, 212.0338,172.0403, 144.0457, 132.0454 uncolored 5.8 negative 575.1223 539.101,449.0882, 423.0769, 407.0779, 327.0521, 289.0725, 287.0548, 285.0419,177.0193, 175.0397, 163.0038, 125.0247 uncolored 4.17 positive 601.1302449.0829, 431.716, 311.0526 uncolored 7.4 positive 496.157 noneuncolored 4.53 positive 291.0866 207.0651, 165.0548, 161.0593 uncolored3.67 positive 318.1545 265.1079, 247.0967, 229.0857, 147.0437, 139.0387,123.0439, 115.0543, 111.0441, 91.0552, 77.0399, 65.0406, 55.0207uncolored 7.4 positive 512.1319 none Uncolored 4.42 positive 865.1955713.1505, 695.1389, 575.1172, 205.0844, 187.0751, 163.0598, 145.0497,127.0387, 121.0653, 85.0299, 77.0401, 69.0351, 57.036, 53.0416 Uncolored3.8 positive 291.0859 207.0643, 179.0682, 165.0539 Uncolored 3.67positive 470.1613 399.0965, 339.0746, 320.1014, 161.0598, 147.0436,139.0388, 123.0439, 119.0485, 115.0544, 111.0438, 91.0554, 77.0391Uncolored 4.53 positive 313.0674 279.0533 Uncolored 4.64 positive575.1019 539.098, 529.134, 279.0533, 261.0269, 251.0664, 219.0314,201.0065, 177.0222, 170.406, 158.9965, 140.9861, 121.0652, 98.9752,77.0409 Uncolored 1.04 positive 365.6434 203.052, 185.0414 Uncolored8.35 positive 471.2209 335.095 Uncolored 3.33 positive 577.1332541.1306, 451.0998, 449.0806 Uncolored 3.66 positive 385.081 339.3446Uncolored 4.56 positive 330.0386 311.4504, 279.0465, 237.0408, 201.0073,175.005, 163.006, 126.969, 110.9749, 98.9766, 68.9664 Uncolored 4.03positive 617.6813 311.0522, 287.0526, 191.0045, 173.019, 160.9945,140.0411, 139.0389 Uncolored 4.53 positive 329.041 190.9962, 172.9939,160.988 Uncolored 7.4 positive 336.107 192.0642, 174.0522, 146.0596,132.9961 Uncolored 5.27 positive 383.1665 221.1129, 128.049 Uncolored3.9 positive 471.1259 None Uncolored 4.8 positive 577.1338 559.1175,451.0739, 435.0754, 409.0917, 301.0684, 289.0726, 275.0703, 271.0583,245.0411, 163.0373, 123.0434 Colored 4.99 negative 603.1596 449.1087,439.1136, 421.0948, 299.0563, 271.0261, 259.0621, 175.04 Colored 4.99negative 623.1428 471.0916, 449.1119, 381.0565, 293.0443, 269.0619,269.0464, 227.0335 colored 3.34 negative 447.1531 315.108, 191.0565,174.9567, 135.0455, 89.0257 colored 4.96 negative 733.2036 581.1564,571.1712, 439.1058, 421.0892, 259.0599 colored 5.18 negative 887.2102725.1714, 449.1034, 394.0628 colored 4.99 negative 691.1336 645.1358,623.1358, 623.1447, 539.0832, 471.0935, 449.1107, 381.0565, 309.0367,293.4312, 269.0458, 225.0515 colored 4.99 negative 601.4094 none colored10.59 positive 334.1114 306.1059, 230.0734, 229.0682 colored 5.01positive 625.6052 473.1048, 311.0514, 203.0624, 127.0308, 126.0243,105.0458, 77.0403, 58.9978, 51.0265 colored 5.01 positive 603.169451.1201, 441.1167, 395.1102, 289.0697, 271.0589, 243.0636, 215.0697,147.0432

Structure Elucidation

Purifying the extract consisted of multiple chromatographic steps. As iscustomary with natural products, changes in the LC profile whereencountered between seed batches. The initial purification step wasfiltration and purification with amberlite, leading to a redder extract(˜29% yield). The semi-pure, post-amberlite extract was then purifiedusing a preparatory C18 HPLC column (FIG. 17). A single fraction fromthat analysis, F12, was further purified using a Restek ultra aromaxpreparatory HPLC column (FIG. 18). The compound eluted as the second oftwo overlapping compounds. The pure F12 sample, collected from the ultraaromax column, was analyzed via high resolution MS/MS. F12 was found tobe a yellow solid. Total concentration in seed extract was notcalculated due to the limited quantity, but it is believed to be in thelow PPB range. HRMS calculated molecular formula for 603.1675 the peakwas C₂₉H₃₁O₁₄. An abundant m/z 441.1160 fragment (Δm/z 162), indicatedthe presence of a glucose moiety (FIG. 19). After passing through 3-6purification steps, the extract still retained some impurities includinganother [M+H]⁺ 603.1687 compound, an [M+H]⁺ 917.2639 compound containingan m/z 603 moiety, and finally an [M+H]⁺ 1205 dimer produced from thecombination of two [M+H]⁺ 603 compounds (FIG. 20). Full MS data for thepurified compound F12 are listed below. The spectrum is shown in FIG.19. The MS m/z (relative intensity) was 603.1675 (42.1%), 441.1160(74.4%), 289.0695 (100%), 243.0641 (15.2%), 215.0682 (7.3%), 123.0436(4.9%). Attenuated Total Reflectance Fourier Transform Infraredspectroscopy (ATR-FTIR) analysis showed a broad OH band at 3300 cm⁻¹ anda peak at 1740 cm⁻¹ indicating the presence of a C═O stretch (FIG. 21).The full ATR data for purified F12 is as follows. IR (cm⁻¹): 3300, 2900,1740, 1640, 1580, 1475, 1350, 1300, 1200, 1120, 1100, 1030, 850, 800,650, 550, 500. Based on the above data, as well as high resolution NMRanalysis, the compound was found to be a glycosylated benzotropolonewith multiple side chains, having a molecular formula of C₂₉H₃₀O₁₄ (FIG.22). A summary of the NMR data for F12 can be seen in Table 3. Spectrafrom samples in (CD₃)₂SO can be seen in FIG. 23 through FIG. 28.

TABLE 3 NMR Correlations of F12 in (CD₃)₂SO Position C ppm H ppm HMBCCOSY  1* 28.9362 2.777, 2.647 6, 16  2* 43.8557 2.099, 1.943 5  3*50.2423 3.523, 3.338  4* 61.442 3.639, 3.400 29  5* 64.1187 3.782, 3.3642  6* 64.3734 4.1 1  7* 70.3375 3 10  8* 73.8364 2.86 10 9 76.8902 4.474 10* 77.2339 3.05 7, 8 11* 79.9933 5.09 12  88.9941  2, 17 13* 90.7046.14 14, 25 14  102.7507 13 15  103.2829  1 16* 103.331 4.01 1 17*113.1622 6.49 18* 115.1565 6.94 19* 115.3991 6.75 20* 118.3498 6.73 1821  130.1123 19, 20 22  145.2667 8.91 19, 20 23  145.3811 8.91 18 24 155.383 25  165.3164 13 26  166.5427  3 27  172.4969 28  178.3405 29 192.7777  4

The full data for F12 in (CD₃)₂SO are listed below. Additionalexperiments were conducted on F12 dissolved in D₂O, however it wasprimarily the (CD₃)₂SO data that were used in assigning positions in thestructure. Data of F12 in D₂O can be found in FIG. 30 through FIG. 36.

Due to structurally similar impurities in F12, some additional peaks areexpected and may be due to these contaminates. The ¹H NMR spectra (FIG.23) (500 MHz, (CD₃)₂SO) shows δ 8.91 (s, 2H), 6.94 (s, 1H), 6.74 (dd,2H), 6.48 (s, 1H), 6.14 (s, 1H), 5.08 (s, 1H), 4.90 (d, J=15.5 Hz, 1H),4.14-4.06 (m, 2H), 4.02 (d, J=7.7 Hz, 1H), 3.78 (td, J=9.3, 6.1 Hz, 1H),3.63 (d, J=11.7 Hz, 1H), 3.51 (d, J=14.6 Hz, 1H), 3.33 (m, 3H), 3.03(dp, J=25.5, 9.3, 8.9 Hz, 1H), 2.86 (t, J=8.3 Hz, 1H), 2.77 (dd, J=16.6,4.0 Hz, 1H), 2.64 (d, J=16.0 Hz, 1H), 2.10 (ddd, J=14.5, 8.6, 5.7 Hz,1H), 1.91 (m, 1H).The proton spectrum (FIG. 23) showed many multipletsin the 5-2 ppm region, indicative of sugar protons and OH groups. Thereare five CH₂ groups, however, for those groups on carbons 3, 4, and 5,one proton signal from each CH₂ group is hidden by a large water peak atapproximately 3 ppm, making them difficult to identify. The spectrumalso showed a very broad, low intensity peak near 11 ppm. This peak islikely to be due to the interaction between the OH on C26 and the C═O atC29. These HO—O═C correlations have been observed in similar compounds,often appearing very downfield around 10-13 ppm. It was necessary to useof (CD₃)₂SO and D₂O as solvents due to low solubility of F12 in anyorganic solvent. Unfortunately these solvents cause some difficultieswhen trying to identify CH₂ protons, and the many OH groups whichpossess protons which exchange rapidly in these solvents, leading to adecrease in the intensity of their signals.

In the carbon spectrum (FIG. 24), 30 peaks were found, 29 of which wereassigned to F12. Assigned peaks are marked. Specifically, the peaks areassigned as follows δ 192.76 (C═O, C29), 178.33 (C═O, C28), 172.48 (C═O,C27), 166.53 (C26), 165.30 (C25), 155.37 (C24), 145.37 (C23), 145.25(C22), 130.10 (C21), 118.34 (CH, C20), 115.39 (CH, C19), 115.15 (CH,C18), 113.16 (CH, C17), 103.32 (CH, C16), 103.28 (C15), 102.74 (CH,C14), 90.70 (CH, C13), 88.99 (C12), 79.99 (CH, C11), 77.23 (CH, C10),76.89 (CH, C9), 73.83 (CH, C8), 70.33 (CH, C7), 64.37 (CH, C6), 64.11(CH2, C5), 61.44 (CH2, C4), 50.24 (CH2, C3), 43.85 (CH2, C2), 28.94(CH2, C1). A carbon at 113.16 ppm had a broad signal of very lowintensity, possibly because of a short T₂ relaxation time, however,correlations in both the DEPT-edited-HSQC as well as in the HMBC, provethat it was a true peak, the carbon of which belonged to F12.DEPT-edited-HSQC confirmed sixteen carbon-hydrogen connections,including the presence of five CH₂ groups, indicated by red(negative)signals in FIG. 25.

HMBC spectrum correlations, indicated by arrows, are shown in FIG. 26. Acorrelation between carbon 29 and 4 may indicate the presence of theglucose moiety on the tropolone ring, while a correlation between carbon26 and 3 may indicate the presence of another, isolated CH₂ on thearomatic ring.

COSY correlations (FIG. 27) were crucial in determining the presence oftwo adjacent CH₂ groups on carbons 2 and 5. It also indicated thepresence of a separate, non-aromatic ring spin-system around carbons 6,1, and 16. The lack of a COSY correlation to carbon 3 indicated itsisolation from adjacent protons, while a single COSY correlation betweencarbon 4 and 9 indicating that it was the CH₂ of the glucose moiety.TOCSY correlations indicated connections between protons of the glucosemoiety. A correlation between carbon 18, 19, and 20 indicated theirclose proximity on the benzotropolone moiety.

F12 as a Food Colorant and Synthesis of F12

In the stability study, CASE proved to be a relatively stable coloranteven over a variety of light and temperature conditions. As it is watersoluble, CASE lends itself particularly to beverage and candy uses, butwould also do well as a component of a flavor or sauce mix. For bakingpurposes, CASE provides a rich, heat stable color which is a commonconcern when working with natural colorants. However, special care mustbe taken when considering the final texture and mouthfeel of CASEcolored food products, as high concentrations may lead to a denser crumbtexture, or an antioxidant related decrease in maillard browning (Dabaset al., 2011. J. Food Sci 76:C1335-41; Dabas, 2012, Ph.D. Thesis, ThePennsylvania State University). For other food products such asfrostings and fillings, it may be possible to combine CASE with aluminaor some other material to create a lake. The use of semi-pure CASE infood products is possible; however it is necessary to add aconcentration 10-100 times more CASE than the corresponding amount ofartificial colorant needed to produce a similar color. This is due tothe low concentration of colored compounds within the extract. F12 isbelieved to be particularly potent, as it produces a vibrant color inthe seed extract despite its presence in the low PPB range. It will beimportant to assure the safety of CASE consumption before production offoods with added CASE, especially those with relatively large amounts ofsemi-pure CASE.

Synthetic production of F12 is likely to become a more efficient methodof acquiring the compound than the extensive time and materials neededto purify the compound directly from seeds. F12 will be able to producea wide range of colors from pale yellow, to orange, to red, to a deepred-brown color. This wide range of colors is achievable by placing thecompound under alkali conditions before readjusting to the desired pH.This means that even in low pH foods CASE can provide a range of yellowand orange colors, which is ideal for its use in beverages juices andsodas, as well as the many unique fall or Halloween treats such asorange colored milk.

Preparation of Comparison of colored and uncolored seed extracts showeda number of known compounds as well as some of undetermined structure.Some of these compounds may act as precursors for F12, however, due tothe low amount needed for complete formation of F12, it is unlikely thata change in the overall concentration of those precursors would beobserved. Preparation of an uncolored extract proved to be particularlydifficult, as even immediate addition of the seeds to a tropolonesolution led to a slightly yellow extract. Once a seed is cut or damagedin anyway, it immediately begins forming the orange compounds. For thatreason completely colorless extract was unable to be produced.

Purification of F12 from avocado seeds proved to be a very time andresource intensive process with many steps. The inhibition of colorformation caused by the addition of tropolone implied the likelihood ofa benzotropolone moiety in the colored compound. High resolution massspectrometry, as well as NMR analysis, confirmed the presence of aglucose moiety which is believed to be the cause of the compound's lowsolubility in organic solvents. ATR confirmed the presence of C═O bonds,and specifically the presence of a carboxylic acid. The presence of anaromatic ring-CH₂-carboxylic acid system was confirmed through acombination of NMR experiments.

F12 was indeed found to be a novel glycosylated benzotropolone compound.COSY and TOCSY experiments confirmed the presence of another spinsystem, removed from the benzotropolone moiety which was found to be aring-fused butenolide moiety (FIG. 29), similar to that found inbuttercups, or the crow's foot family (Ranunculaceae) (Guerriero andPietra, 1984, Phytochemistry 23:2394-6). An initial synthesis attemptcould make use tyrosinase from mushrooms or horseradish peroxidase toprovide the enzymatic formation of the 7-membered ring. Compounds suchas benzenacetic acid and 2-(3,4,5-Trihydroxyphenyl)ethanol (FIG. 29)could combine via enzymatic synthesis to form the benzotropolone moietyincluding the CH₂—COOH on the aromatic ring, as well as —OH groups whichcould easily be involved in the addition of multiple side chainsincluding a glucose moiety and a fused-ring butenolide moiety.

Esters can hydrolyze via a variety of mechanisms, particularly in thecase of lactones. In the presence of a strong base lactones canhydrolyze to form their parent compound, a bifunctional straight chaincompound (Gomez-Bombarelli et al., 2013, J Org Chem 78:6880-9). Thecolor dependence of F12 on pH may be due to the deprotonation of variousOH and CH₂ groups, and the opening of the butenolide rings at high pH.Ring opening and the deprotonation of OH and CH₂ groups could lead to anincrease in the number of double bonds in the compound, causing anincrease in conjugation which is observed as a red-shift in the colorspectrum.

Effect of Semi-Pure CASE on Viability of Human Cancer Cells

Previous studies have shown the anti-cancer effects of some coloredavocado seed extracts in LNCaP human prostate cancer cell lines (Dabaset al., 2011. J. Food Sci 76:C1335-41; Dabas, 2012, Ph.D. Thesis, ThePennsylvania State University). Following the same protocol, the effectof semi-pure CASE on cell viability was determined using the MTT assay.The Concentrations of semi-pure CASE used were 0, 1, 3, 5, 10, 15, 20,25, and 30 μg/mL. In brief, cells were seeded (104 cells/well) in 96well plates and allowed to attach overnight. The cells were treated withCASE for 6, 12, 24, and 48 h. After CASE treatment, cells were combinedwith MTT and absorbance read at 540 nm. FIG. 37 shows the results ofthis experiment. In this study, semi-pure case did not show any decreasein the viability of LN-CaP cells over 48 hours. In some cases, anon-significant trend of increasing cell viability with increasingsemi-pure CASE was observed, which could be due to the high polyphenoliccontent of the extract.

Compounds Isolated From Colored Avocado Seed Extract as Natural Colorant

A colored avocado seed extract is shown herein to be relatively heat,light, and shelf stable and was able to produce a variety of yellow,orange, and red colors. This extract may confer some positive healthbenefits due to the antioxidant activity associated with its highpolyphenol content. While a semi-pure extract may be useful in someapplications, the high concentration needed may prove to be a hindrancefor its use in foods. In the future, a synthetic route for theproduction of F12 will make it possible to expand its uses as a naturalcolorant. Before that time some other studies will need to be conductedas well, in order to determine the safety of consumption of thesemi-pure extract and F12, and to help determine an ADI for consumers.

The whole extract presents as a dark or reddish orange color, while F12is a yellow orange. Further analysis of the whole extract couldpotentially determine the source of the redder color, which is ofparticular interest to the natural color market. In previous work, CASEwas shown to have some beneficial anti-cancer, anti-inflammatory, andanti-oxidant properties when tested in vitro in human cancer cell lines(Dabas et al., 2011. J. Food Sci 76:C1335-41; Dabas, 2012, Ph.D. Thesis,The Pennsylvania State University). Those effects were not able to bereplicated using the semi-pure CASE. This indicates that the coloredcompounds are likely not solely responsible for the health beneficialeffects observed in cell line studies. It is more likely that otherpolyphenol compounds in the extract are responsible for the effectsobserved in cell line studies. Further analysis of whole extracts andextracts at various levels of purification, such as the principalcomponent analysis that was conducted on the colored and uncoloredextracts in this project, may be useful for aiding in the determinationof which compounds are responsible for those effects.

Example 2 Modifications and Derivatives of F12

A polyphenol oxidase (PPO) catalyzed reaction produced the primarypigment in this extract, F12, which is a novel glycosylatedbenzotropolone compound with carboxylic acid and fused-ring butenolidecontaining side chains. Though the color is stable at room temperature,liquid chromatography-mass spectrometry (LC-MS) indicates that theindividual compounds may not be stable, forming dimers and othercompounds in aqueous solution. The most abundant colored fraction showedF12 to have an ion [M+H]⁺ with m/z 603.1675 in positive mode. Based onthe presence of an abundant m/z 441 fragment (Δm/z 162), it ishypothesized that this compound is a glycosylated benzotropolonecompound. However, the same extract also contained other [M+H]⁺ ionsincluding a m/z 603.1687 compound, a m/z 917.2639 compound with a m/z603 moiety, and finally an m/z 1205 dimer produced from the combinationof two m/z 603 compounds.

F12 (1), isolated from avocado seeds, is a glycosylated benzotropolonecompound with a fused-ring butenolide moiety. When treated with base,the lactone ring of the fused-ring butenolide may open to form 2. Manyvariations of 1 may be formed in the avocado seed through substitutionof the R groups (3) with different compounds present in the seed. Eachof which may be useful as a food colorant (FIG. 38).

The fused-ring butenolide may be replaced with some aromatic alcoholwith an unsaturated side chain of any length (4). The glucose moiety maybe changed to a perseitol/D-mannoheptulose moiety, or other mono, di, ortrisaccharide (5-7). Additionally, the carboxylic acid group could bechanged even by extending an alkene chain before the carboxylic acid(8). An aglycone compound (9) is possible and may have improvedsolubility in lipid systems. F12 may also be able to dimerize withitself (10) or other compounds.

Example 3 Perseoranjin and Derivatives of Perseoranjin as a Colorant

The experimental results described herein optimize extraction protocoland structural analyses, and studies the steps of the formation of thecolor.

The materials and methods employed in these experiments are nowdescribed.

Preparation of Extracts

Seeds were weighed and cut with a knife. A weight of deionized water(DI) equivalent to 8 times the weight of seeds was added to the seeds ina Waring blender and crushed for 90 s at high speed. A timer was thenstarted. The resulted paste was kept in the blender until t=10 min. Itwas then filtrated on Whatman paper filter grade 4 of 110 mm diameter.The solution collected from filtration was left at room temperatureuntil t=30 min (FIG. 39).

This resulting solution was then placed on a resin into a chromatographycolumn and then eluted with ethanol. Both solutions (after filtrationand after chromatography) were used to carry out several measures.

Solid Yield Determination

After filtration, the solution obtained and the solid on paper filterwere weighed separately. An extract of each was placed in oven at 50° C.until they were completely dried. Then they were weighed and the solidyield was calculated (per gram of seed). This experiment was performedon 3 different seeds. The solid yield was calculated afterchromatography. The filtrated solution was placed in the chromatographycolumn. The solution eluted with ethanol was then dried and weighed.

Measure of Absorbance

Visible absorbance spectra were recorded after 30 min of reactions(λ=400 nm to 600 nm) using an Agilent 8453 spectrophotometer (AgilentTechnologies, Santa Clara, Calif., U.S.A.) by placing samples indisposable 1.5 mL cuvettes (Plastibrand, Wertheim, Germany).

Enzymatic Activity Assay

This test evaluated the difference of enzymatic activity depending onthe temperature of reaction (24° C., 30° C., 40° C.). The enzymaticactivity was assessed with the absorbance after 30 min of reaction.Seeds were blended in 0.1M sodium phosphate buffer. The weight of bufferwas 8 times the weight of seed.

The use of a buffer allows for three different steady pH for eachtemperature of reaction: 5.8, 6.9, 7.9. These pH are chosen because itseems that the optimum pH for PPO is in the interval [5.5-8] dependingon the fruit or vegetable (Nagodawithana T, Reed G 1993).

There are thus 9 combinations pH-temperature. Each of them was repeatedthree times to reduce the variability due to the seed.

Impact of pH on Color Formation

To evaluate the impact of pH modification on color formation differentvolumes of NaOH ₂N and HCl 1N were added to several samples from thesame seed. Hydrochloric acid was added by 0.02 mL reach a pH of about 2.Sodium hydroxide was added by 0.01 mL to reach a pH of about 10. Theoptical density was then measured at t=30 min.

Determination of Acid pK_(a)

Previous research suggests that there is a carboxylic acid group in thestructure of the pigment studied. To evaluate the pK_(a) of this acid insolution a purified extract (after chromatography) was used. Ethanol wasremoved in oven and the recovered solid was diluted in DI water. Theresulting solution was titrated with NaOH 0.25N.

Study of the Precipitate

A precipitate formed after incubation overnight at room temperature. Asa difference of precipitate color was observed depending on pH, severalextracts were prepared by addition of NaOH 2N or HCl 1N to evaluate moreprecisely the impact of pH on this agglomerate.

In order to avoid the formation of the precipitate, others extracts wererealized. After filtration on Whatman paper grade 4 (retention ofparticles of 20-25 μm), the resulting solution was either centrifuged orboiled or filtrated again on Whatman paper grade 2 (retention ofparticles of 8 μm). The supernatant of centrifugation was stored at roomtemperature and the formation of a precipitate was watched. The boiledsolution was filtrated again on Whatman paper grade 4, because some masswere formed, and stored at room temperature. These different processeswere all realized from the same seed.

The results of the experiments are now described.

Solid Yield Determination

The mean solid yield obtained from the liquid part after filtration was8.1%. The mean solid yield obtained from the solid part was 42.9%indication that the protocol design allows us to recover 51% of theavocado seed solid. The seed moisture content is about 50% (Olaeta J Aet al. 2007), and therefore the extraction protocol did not result inloss of solid.

Measure of Absorbance

The absorbance spectrum between 400 nm and 600 nm is generally the samefor all seeds. The absorbance peak is about 475 nm (FIG. 40).

Relation Between Seeds Weight and Absorbance

A test was carried out on 30 seeds to assess the presence of a linkbetween seed weight and pigment concentration. The interest of a linkbetween these two parameters is that it might allow to standardize theextraction protocol, and to optimize it according to the weight of theseed.

The test indicates that there is no link between the weight of seeds andthe pigment concentration. Statistical analysis was performed todemonstrate this. The amount of pigment may be linked to the degree ofmaturity of the seed rather than to its size. It would be interesting todetermine the evolution of the amount of pigment during development ofthe seed.

The absence of link between weight of seed and the absorbance may be anadvantage because it would allow for the use of a seed regardless of itsweight.

Evaluation of the Impact of Temperature on Enzyme Activity

The mean absorbance peak from extract obtained at 35° C. and 45° C. weredifferent from the absorbance peak from extract obtained at 24° C. (roomtemperature) regardless of the pH of the buffer used (FIG. 41). However,the test was not performed on a sufficient number of seeds to evaluatethe significance of the results. If we consider the test as significant,the interest of carrying out the protocol at 45° C. has to be verified.Thus, the stability of absorbance (which reflects color stability)during 3 days will be evaluated either for samples extracted at 24° C.and those extracted at 45° C.

Study of the Absorbance Stability

The absorbance peak of 13 seeds (a first batch of 7 seeds and a secondbatch with 3 seeds at 24° C. and 3 at 48° C.) was measured once a dayfor 3 days, on solutions kept at room temperature. The absorbance peakdiminished over the course of a couple of days before stabilizing at thesame value regardless of the temperature (FIG. 42). Carrying out thetest at 45° C. instead of the room temperature is thus not beneficial.The evolution of color is more interesting to evaluate on the solutionobtained after chromatography than as to evaluate most purified extractthat we can.

Impact of pH on Color Formation

The color formed during the reaction depends on the pH of the solution.The solution varies from a yellow color at low pH to brown/red color athigh pH (FIG. 43). When the opposite experiment was carried out thecolor was not recovered. Indeed when a solution at pH 11 was acidifiedto pH 2, the color was dark orange. (FIG. 44). And when a solution at pH2 was brought to pH 11, the color was not brown/red. This indicates thatthe color change with pH was not reversible. This result suggests thatthe pH of the food is not insignificant and must be taken intoconsideration if the extract is used as food colors.

Another aspect which deserves to be studied was the kinetics of colorchange at pH 11. When the solution was just prepared, it was stillorange but some time later it turned to dark red. A solution was thenprepared at pH 11 at its absorbance spectrum was recorded every 30 min.The whole spectrum was interesting but the evolution of the absorbanceat the wave length corresponding to a red color of the solution (k=418nm is chosen) was the most important to observe. Indeed, it is worthknowing how much time is needed for the color to stabilize after achange of pH. After about 16 hours, the absorbance at 418 nm remainedconstant (FIG. 45).

Determination of Acid pKa

Titration has resulted in an approximation of the pKa of the acidpresent in the structure of the pigment by the tangent method. The pKabelongs to the interval [5.8-6.2] (FIG. 46). Knowing this pKa will helpin the determination of the exact structure of the pigment. It will alsohelp to understand the behavior of the pigment according to theenvironmental conditions.

Study of Storage Condition of Seeds Before Reaction

In a perspective of commercialization, most avocado pits will be sentfrom California or Mexico. It is difficult to send fresh seeds on whichit would remain avocado flesh, which could mold during the journey. Thiswill test if it is possible to mix several non fresh seeds (boiled,frozen . . . in which the enzymes, among them that responsible for thepigment formation, will be denatured) with one fresh seed (the only oneto bring enzyme).

The test was carried out by mixing 2 seeds (2 fresh or 2 boiled or 1boiled with 1 fresh). Then absorbance peaks were compared. Every batchof two seeds was repeated three times. By observing the samples, itappears as if there was a difference in color between the threesolutions (FIG. 47). But comparison of the absorbance between two freshseeds and mixture fresh/boiled showed a non significant difference (FIG.48).

Although there was no absorbance difference, boiling the seeds appearedto cause gelatinization of starch they contain, which may beproblematic. Indeed solutions with two boiled seeds or mixed solutionswere very difficult to filter. This step was extremely time-consuming,much more than with only fresh seeds, and it was not very effective.These differences were very significant, which was a real problem in aperspective of industrialization.

Study of the Precipitate

Avocolor™ has also been tested. The main problem they shared is thepresence of a precipitate that forms over time when they re-dissolve thepowder in an aqueous solution. This precipitate is an agglomerate and isgooey and gelatinous, often light colored which deposits on the samplebottom. The precipitate grows with time. Sometimes after few days, itascends in the sample to form a mass in the middle of the liquid. Whenthe precipitate was removed from a sample, it was reformed few daysafter.

When the solution obtained after filtration was centrifuged or boiled, aprecipitate was formed but after several days (3-4 days vs 1-2 daysnormally) and it was really small. The second filtration with Whatmanpaper grade 2 did not seem to impact the precipitate form, color orquantity. Therefore, there may be an effect of pH on the precipitate.The precipitate were recovered from samples and then put in acidifiedethanol. At high pH the precipitate was bright orange/red whereas at lowpH it was light orange/uncolored (FIG. 49 and FIG. 50). Moreover, at lowpH the precipitate looked like filaments or small balls whereas theprecipitate at high pH had a cloudy appearance.

Since the avocado pit contains starch (it is an amylaceous seed), thismolecule might be a compound of the precipitate. The avocado seed alsocontain pectin (Pahua-Ramos et al. 2012). It could explain the differenttypes of precipitate depending on pH. Pectin tends to jellify at low pH(around pH 3) whereas it is unstable at higher pH.

Alternatively, if the solution after filtration contains protein theremay be, depending on the conditions, the formation of a complex with thepolyphenols. The complex formed with protein and polyphenols is oftendue to the presence of Van der Waals interaction. This bound is thusreversible. If such a link is formed, it results in a decrease of theelectrical charge of the protein and an increase of its molecular mass.Then the complex flocculates. (Moreno, Peinado 2012). This binding ispH-dependent. Indeed pH changes the electrical charge of the protein andof the polyphenols. When the pH increases the electrical charge ofpolyphenols becomes more negative. Beyond the isoelectric point theprotein charge is negative. This could explain that the precipitate doesnot look like the same depending on pH. At certain pH there is maybe aflocculation of protein-polyphenol complex whereas at other pH theprecipitate could be due to another phenomenon. The interest ofchromatography would then be to remove the protein and starch, and so toreduce the precipitate formation possibilities.

This work shows many aspects of the avocado seed which have beenstudied. The solid yield was calculated. The impact of severalparameters on the absorbance was evaluated: seed weight, temperature,pH, seed storage conditions. It appears that pH is a key factor tounderstand the phenomenon that occurs. pH affects both the colorintensity, the absorption spectrum of the extracts, but also theprecipitate which forms. This precipitate is still the major barrier tothe use of the Avocolor™ in food. First safety tests have been performedon mice. There were no signs of diseases. But other studies are neededto know the extent of food applications of this extract.

Example 4 The Effect of Sodium Hydroxide Treatment on the Color of“Perseoranjin” Via Absorbance Measurement

A basic solution was prepared for absorbance measurement. A 0.1% avocadoextract was generated by diluting 10% avocado extract with deionizedwater . A 0.05% avocado extract was generated by diluting previous 0.1%avocado extract with 0.01 M NaOH. The 0.05% avocado extract was obtainedwith outset absorbance below 1 and pH 10-12. The Absorbance measurementof 0.05% basic avocado extract at time zero and followed by every 30mins for 8 hours at pH 10.58, 11.44, and 11.40 (FIG. 53).

Absorbance of BAEs increases as time exceeds and observed color of thesolution shifts from light yellow to orange. BAEs appeared orange due totheir very low concentration of avocado extract (0.05%), however theyactually are mixture of colors according to the UV spectrums. In thebeginning of all BAEs spectrums, wavelengths of blue and green aregreatly absorbed resulting in a red and orange mixture color of thebasic avocado seed extract.

For example once the measurement was done at 90 mins according to FIG.54, another peak gradually appears absorbing in the violet region. Thus,the extract has a present color mixture of yellow, orange and red after5 hrs of base addition.

Absorbance measurement at time zero (comparison of absorbance at 400 nm)of experiment #3 was more delayed than experiments #1 and #2 creating awide gap between graphs 0 mins and 30 mins of FIG. 5. This leads toabsence of some important transition peaks. Experiment #5 below alsogave similar results due to delay of measurement.

The maximum wavelengths observed were then examined separately. For bothwavelengths, absorbance increases as a logarithmic function.

Addition of NaOH to avocado extract causes an abrupt increase ofabsorbance in the first 100 mins and slowly decelerates as it reachesminute 200, while becoming fairly constant as it arrives at minute 300and so on as observed in FIG. 53, FIG. 54, and FIG. 58 above. Thus,following repetitive experiments were simply done for 5 hours or 300mins in total.

A neutral solution was also prepared for absorbance measurement. BAE #5was further used immediately after 5 hours to alter its pH from 11.35 toapproximately pH 6 by addition of 0.1 M HCl. After absorbancemeasurement (5 hours), BAE #5 was pH 10.41; HCl before addition was pH0.72. Table 4 shows the pH titration.

TABLE 4 Condition pH Drop 1 9.85 Drop 2 9.23 Drops 3, 4 6.90 Drop 5 and6.26 stir 2 mins Drop 6 and 5.35 stir 2 mins 5 mins of 5.21 stirring

6 drops of HCl effectively neutralized BAE #5 according to pH value,however absorbance of NAE continued to increase as time exceeds, whichcan conclude that NaOH was not successfully removed. Orange precipitateswere observed on dayl of absorbance measurement pipette was used todisperse them before analysis (FIG. 55). Graphs for day2_1 and day2_2are inaccurate due to uneven dispersion of colored precipitates (FIG.56).

The precipitates of neutral avocado extract was tested for solubility.Solubility of the precipitates was tested with ethyl acetate andmethanol. Both solvents were not able to remove them from the filter.Thus, precipitates did dissolve in neither ethyl acetate nor methanol.

Normal, basic and base washed avocado extracts were compared. Normalavocado extracts were prepared by generating a 0.05% avocado extract bydiluting 10% avocado extract with deionized water and then itsabsorbance was measured. Basic avocado extracts were prepared bygenerating a 0.05% avocado extract by diluting 10% avocado extract withdeionized water and then its absorbance was measured then its absorbancewas measured at time zero and 300 mins. Base washed avocado extractswere generated by adding basic avocado extract (after 5 hrs) was thenadded to resin, which adsorbed the color pigments followed by washingthem off by acid/EtOH and measuring absorbance of the final obtainedsolution. However, the solution that was only vacuum filtered containedpulverized resin from stirring. Thus, some of the cloudy yellow solutionwas filtered once again with a syringe filter and obtained a clearyellow solution. Both solutions were analyzed for their absorbance (FIG.57).

The absorbance patterns of normal avocado extract (norm) and filteredbase washed avocado extract (FBW) are very similar to one another. Thus,it could be concluded that NaOH was successfully removed from the finalsolution (FIG. 58).

Example 5 The Stability of Perseoranjin in the Presence of ChemicalsCommonly Added to Foods Under Common Storage Conditions

The results presented herein describe the stability of perseoranjin inchemicals.

The materials and methods employed in the experiments presented in thisExample are now described.

Stability of perseoranjin in Ascorbic acid (Vitamin C) and Potassiummetabisulfite as representative of Sulfur dioxide at 32° C.

0.25% Avocado seed extract solution was added into Ascorbic acid in therange of 0-20 mg/mL and was added into Potassium metabisulfite in therange of 0, 25, 50, 75 and 100 ppm of Sulfur dioxide. The 0, 0.05, 0.1and 0.2 g of Ascorbic acid in 10 mL of 0.25% Avocado seed extractsolution and the 0, 4, 8, 12, 16 mg of Potassium metabisulfite in 10 mLof 0.25% Avocado seed extract solution were measured pH and absorbancein the visible range (400-600 nm) by using pH meter and uv-visspectrophotometer once a week and were incubated at about 32° C.

Stability of perseoranjin in the presence of Proteins by using Gelatin,Casein, and Cherry flavoring as representative of Benzaldehyde at 4° C.

0.25% Avocado seed extract solution was added into 0.1% Gelatin, 2%Casein which was stirred at 55° C. for 10 mins to better Caseindissolvable and 0.2% Cherry flavoring. The 0.01 g of Gelatin in 10 mL of0.25% Avocado seed extract solution, the 0.2 g of Casein in 10 mL of0.25% Avocado seed extract solution and the 0.02 mL of Cherry flavoringin the solution of 9.73 mL of deionized water and 0.25 mL of 10% avocadoseed extract solution were measured pH and absorbance in the visiblerange (400-600 nm) by using pH meter and uv-vis spectrophotometer once aweek and were kept refrigerated (4° C.).

The results of the experiments presented in this Example are nowdescribed.

Thermal Stability Test of Avocolor in Presence of Ascorbic Acid at 32°C.

Amounts of Ascorbic acid or Vitamin C including 0, 0.05, 0.1 and 0.2g=were in the range of 0-20 mg/mL (0, 5, 10, 20 mg/mL) were added into10 mL of 1% Avocolor solution (1 ml of 10% Avocolor solution and 9 mL ofdeionized water) These concentrate ratio solution gave too high colorintensity, the absorbance peaks exceeded one. Thus, the highconcentration were diluted to ¼ or 0.25% Avocolor solution (used 0.25 mLsolution and 0.75 mL deionized water) before absorbance measurement.

The wavelengths of indigo and blue were absorbed resulting inorange-yellowish color but all baseline solutions did not diluted to ¹/₄in absorbance measurement, so reference solution (Ascorbic acid indeionized water) did not diluted as Ascorbic acid in Avocolor solutionwhen the absorbance peaks were measured day 0.

The color of reference solution used in absorbance measurement wastranslucent colorless, then reference solution turned yellow after oneweek and got darker yellowish color over time caused by a browningreaction of Ascorbic acid oxidation as its reacted with oxygen whichaffected from the heat at 32° C. in incubator. The color of Ascorbicacid added Avocolor solution was lighter yellow-orangish than thestandard solution (Avocolor solution) at time zero and then got darkerorangish over time due to the Ascorbic acid oxidation reaction becausethe wavelengths of indigo and blue were more absorbed over the timeresulting in the absorbance peak of Avocolor solution added Ascorbicacid increased (FIG. 59 and FIG. 60).

These experiments were repeated a second time (FIG. 61 and FIG. 62).Table 5 shows the pH of different concentrations of Ascorbic acid (AA)in 1% Avocado seed extract solution in 2 weeks.

TABLE 5 Day 0 Week 1 Week 2 0 g AA in 1% Avocolor 4.39 4.11 4.35 0.05 gAA in 1% Avocolor 3.07 2.01 2.20 0.1 g AA in 1% Avocolor 2.87 1.96 2.040.2 g AA in 1% Avocolor 2.72 1.82 1.93

The Avocolor solution that added Ascorbic acid turned dark orange whichwas similar to the standard solution but the highest concentration gotdarker orange than the standard solution. The reference solution colorwhich Ascorbic acid added turned darker yellowish from week 1 by abrowning reaction of Ascorbic acid oxidation as its reacted with oxygenwhich affected from incubator's heat at 32° C. Moreover, the referencesolution of 0.2 g Ascorbic acid added still was the lightest yellowishreference solution.

The wavelengths of indigo and blue were absorbed more than week 1resulting in the absorbance peak of the standard solution and 0.5 gAscorbic acid added solution increased due to the color turned darkerorange. The absorbance peak of Avocolor solution that 0.1 and 0.2 gAscorbic acid added were different from others due to the pH changed andcolor changed from Ascorbic acid oxidation reaction.

Avocolor solution which Ascorbic acid added turned darker orangeyellowish over time due to a browning reaction of Ascorbic acidoxidation as its reacted with oxygen which affected from incubated ataround 32° C. but pH of Avocolor solution that Ascorbic acid addeddecreased over one week and increased in week 2 but did not exceed pH attime zero.

Thermal Stability Test of Avocolor in Presence of PotassiumMetabisulfite at 32° C.

Potassium metabisulfite (K₂S₂O₅) in the amounts of 0, 4, 8, 12 and 16 g,which were in the range of 0-100 PPM of Sulfur dioxide, SO₂ (0, 25, 50,75 and 100 PPM) were dissolved in in 0.25% Avocolor (0.25 ml of 10%solution and 9.75 mL of deionized water).

The Avocolor solution that added Potassium metabisulfite turned gentlylighter orangish color from the standard solution (Avocolor solution) attime zero because the wavelengths of indigo and blue of Potassiummetabisulfite added were less absorbed than the standard solution. Theabsorbance peaks were gently decreased due to the higher concentrationof Potassium metabisulfite added. The different concentrations ofAvocolor solution that added Potassium metabisulfite approximately hadpH 3.5-4.Table 6 shows the pH of different concentrations of Potassiummetabisulfite in 0.25% Avocolor in 1-2 weeks.

TABLE 6 Experiment 1 Experiment 2 Day 0 Week 1 Day 0 Week 1 Week 2  0 mgK₂S₂O₅ in 0.25% Avocolor 3.93 4.37  4.33 3.76  4.26  4 mg K₂S₂O₅ in0.25% Avocolor 3.95 3.93  4.17 2.50  2.88  8 mg K₂S₂O₅ in 0.25% Avocolor3.93 3.91  4.30 2.96  2.76 12 mg K₂S₂O₅ in 0.25% Avocolor 3.97 3.58 4.32 3.12* 3.68 16 mg K₂S₂O₅ in 0.25% Avocolor 3.94 4.09* 4.30 3.15*3.33

The reference solution color did not change from the beginning. Thecolor of Avocolor solution to which 16 mg Potassium metabisulfite addedwas similar to the standard solution which were darker orangish, 4 mgPotassium metabisulfite added was darker yellowish, 8 mg Potassiummetabisulfite added was the most pale yellow, and 12 mg Potassiummetabisulfite added was the darkest orangish color and was strong smellyin two weeks.

The pH changed did not depend on the concentration of Potassiummetabisulfite added into 0.25% Avocolor solution. In addition, the pHdecreased after one week by incubated at 32° C. and increased after twoweeks but not exceeded pH at time zero besides 8 mg K₂S₂O₅ added whichdecreased over time.

Thermal Stability Test of Avocolor in Presence of Gelatin at 4° C.

The absorbance of 0.1% Gelatin in 0.2 5% Avocado seed extract solutionwas measured over 3 weeks (FIG. 65). The pH of 0.1% Gelatin in 0.25%Avocolor was approximately 4.4-4.5. The wavelengths of blue wereabsorbed resulting in orange mixture of the 0.1% Gelatin in 0.25%Avocolor solution which had three layers; clear orange solution on thetop, muddy orange solution in the middle and orange precipitate at thebottom due to 0.1% Gelatin was precipitated by 0.25% Avocolor. Thesolution of 0.1% Gelatin in 0.25% Avocolor did not changed in 3 weeks.Table 7 shows the change in pH of 1% Gelatin in 0.25% Avocado seedextract solution over 3 weeks.

TABLE 7 Day 0 Week 1 Week 2 Week 3 pH 4.53 4.55 4.50 4.47

The absorbance peak increased in a week because of observing more muddyorange solution and orange precipitate than at time zero but theabsorbance peak decreased in week 2 and week 3 because of losing somecloudy solution and orange precipitate from transferring the solution tocuvette by pipette in absorbance measurement.

Thermal Stability Test of Avocolor in Presence of Casein at 4° C.

The pH of 2% Casein in 0.25% Avocolor was approximately 6-7 in 3 weeks.The wavelengths of green were absorbed resulting in cloudy pink color of2% Casein in 0.25% Avocolor (FIG. 66). Moreover, the cloudy pinksolution did not changed in 3 weeks but the absorbance peaks gentlyincreased over time. Table 8 shows the change in pH of 2% Casein in0.25% Avocado seed extract solution over 3 weeks.

TABLE 8 Day 0 Week 1 Week 2 Week 3 pH 6.42 6.65 6.97 6.84

Thermal Stability Test of Avocolor in Presence of Cherry Flavoring at 4°C.

0.2% Cherry flavoring in 10 mL of 0.25% Avocolor was made by using 0.02mL of Cherry flavoring in 10 mL 0.25% Avocolor (0.25 ml of 10% solutionand 9.73 mL of deionized water).

The pH of 0.2% Cherry flavoring in 0.25% Avocolor was approximately4.3-4.4 in 3 weeks (Table 9). The wavelengths of indigo and blue wereabsorbed resulting in dark yellowish color of 0.2% Cherry flavoring in0.25% Avocolor which did not changed the color in 3 weeks but theabsorbance peaks decreased from time zero and gently decreased tendencyof absorbance over time (FIG. 67).

TABLE 9 Day 0 Week 1 Week 2 Week 3 pH 4.45 4.42 4.45 4.33

Example 6 The Stability and Application of Perseoranjin in Food Matrices

The results presented herein demonstrates the stability of perseoranjinin foods such as yogurt, sprite, corn chips and white chocolate.

The materials and methods employed in the experiments presented in thisExample are now described.

Stability of Perseoranjin in Plain Yogurt

2 mL of 1% Avocado seed extract solution was added into 5 g of plainyogurt compared to 0.2 mL of 10% Avocado seed extract solution which wasadded into 5 g of plain yogurt.

Stability of Perseoranjin in Sprite

1 mL of 1% Avocado seed extract solution was added into 10 mL of Spritecompared to 1 mL of 1% Avocado seed extract solution which was addedinto 10 mL of deionized water. 1 mL of 0.25%, 0.5% and 1% Avocado seedextract solution were added into 10 mL of Sprite and were then measuredthe absorbance in the visible range (400-600 nm) by using uv-visspectrophotometer for 2 days.

Stability of Perseoranjin in Tostitos Corn Chip

One piece of Tostitos was sprinkled by Maltodextrin added Avocado seedextract powder (2.423% Avocado seed extract)

Stability of Perseoranjin in White Chocolate

5 g of white chocolate was melted by incubated at 32° C. for 1 hour andwas then swirled and sprinkled by 0.1 g Maltodextrin added Avocado seedextract powder (2.423% Avocado seed extract)

The results of the experiments presented in this Example are nowdescribed.

Perseoranjin in Plain Yogurt

2 mL of 1% Avocado seed extract solution was added into 5 g of plainyogurt compared to 0.2 mL of 10% Avocado seed extract solution which wasadded into 5 g of plain yogurt resulting in plain yogurt added 2 mL of1% Avocolor was thinner than plain yogurt and plain yogurt added 0.2 mLof 1% Avocolor and was brighter orangish than plain yogurt added 0.2 mLof 10% Avocolor (FIG. 68).

Perseoranjin in Sprite

1 mL of 1% Avocado seed extract solution was added into 10 mL of Spritecompared to 1 mL of 1% Avocado seed extract solution was added into 10mL of deionized water resulting in Sprite added 1% Avocolor solution wasbrighter yellowish than 1% Avocolor solution (FIG. 69).

1 mL of 0.25%, 0.5% and 1% Avocado seed extract solution were added into10 mL of Sprite and were then measured the absorbance in the visiblerange (400-600 nm) by using uv-vis spectrophotometer for 2 days.

The wavelengths of indigo and blue were absorbed resulting in theyellowish solution. The higher concentration of % Avocolor solution, thehigher yellowish color intensity due to the more absorbance peak.Moreover, the yellowish color of % Avocolor solution in 10 mL Sprite didnot changed from the color at time zero (FIG. 70 and FIG. 71).

The absorbance peaks were below 0 due to small bubbles in Sprite thatused as baseline in the absorbance measurement at time zero. The nextday, the absorbance peaks increased from the peaks at time zero andgently increased in Day 2.

Perseoranjin in Corn Chips

One piece of plain uncolored corn chips (Tostitos) was sprinkled byMaltodextrin added Avocado seed extract powder (2.423% Avocado seedextract) resulting in using only 0.3115 g Maltodextrin added Avocadoseed extract powder can be sprinkled on 4.1771 g one piece of Tostitos(FIG. 73).

Perseoranjin in White Chocolate

5.1076 g white chocolate was sprinkled by 0.1002 g Maltodextrin addedAvocado seed extract powder (2.423% Avocado seed extract) and swirled,the orangish chunk came from the Maltodextrin added Avocado seed extractpiece that cannot dissolve in the chocolate at 32° C. (FIG. 74).

Example 7 Structural Determination and Modification of Avocado SeedExtract Perseoranjin

To determine the structure of the avocado seed extract perseoranjin,HSCQ (Table 10), HBMC (Table 11, and COSY (Table 12) were obtained

TABLE 10 HSQC Data^(a) Position δ_(C) δ_(H)  1 103.31 4.02 d (7.7 Hz)  273.84 2.86 brdd.  3 76.89 3.07 brt.  4 70.34 3.00 brt.  5 77.24 3.03 ddd(13, 9, ~1)  6 61.45 3.63 brd. (11.8), 3.40^(b)  1′ 64.12 3.78 ddd(15.1, 9.4, ~1), 3.37^(b)  2′ 43.86 2.10 ddd (14.6, 8.5, 6), 1.93 ddd(14.6, 7.3, ~1)  4′ 50.25 3.52 d, (14.6), 3.33^(b)  7′ 90.71 6.15 s  9′28.94 2.77 dd (16.3, 4), 2.63 d (16.3) 10′ 64.38 4.10 brs. 11′ 80.005.08 s c 113.17 6.50 s e 115.16 6.95 brs. g 118.35 6.72 d (8.3) h 115.406.75 d (8.3) ^(a)In DMSO-d₆. ^(b)Obscured by the water signal.

TABLE 11 HMBC Data^(a) Position δ_(C) δ_(H) Correlations  1 103.33 4.02d 3.78, 2.86  2 73.84 2.86 brdd. 3.07  3 76.89 3.07 brt. 2.86  4 70.343.00 brt. 3.07, 3.03  5 77.24 3.03 ddd 3.00  6 61.45 3.63 brd., 3.40^(b)3.00  1′ 64.12 3.78 ddd, 3.37^(b) 4.02, 2.10, 1.93  2′ 43.86 2.10 ddd,3.78, 3.52, 3.37, 3.33 1.93 ddd  3′ 89.0 — 6.50, 3.52, 3.33, 2.10, 1.93 4′ 50.25 3.52 d, 3.33^(b) 1.93  5′ 166.55 — 3.52, 2.10  6′ 178.38 —3.52  7′ 90.71 6.15 s (none)  8′ 155.41 — 2.63  9′ 28.94 2.77 dd, 2.63 d(none) 10′ 64.38 4.1 brs. 2.63 11′ 80.0 5.08 s 6.95, 6.72, 2.63 12′103.42 6.15 s, 2.77 dd (16.3, 4), 2.63 d (16.3) a 192.78 — 6.50, 3.52,3.33 b 102.75 — 6.50, 6.15 c 113.17 6.50 s (none) d 165.32 — 6.15 e115.16 6.95 brs. (none) f 130.12 — 6.75, 6.72, 5.08 g 118.35 6.72 d6.95, 5.08 h 115.40 6.75 d 6.72, 5.08 i 166.60 — 3.52 j 145.40 — 6.95,6.75 k 145.39 — 6.72 ^(a)In DMSO-d₆. ^(b)Obscured by the water signal.

TABLE 12 COSY Data^(a) Position δ_(H) Correlations^(b)  1 4.02 d (7.7)2.86 (s)  2 2.86 brdd. (9, 4.75 (OH) (m), 4.02 (s), 3.07 (s) 7.7)  33.07 brt. (9) 4.89 (OH) (w), 3.00 (s), 2.86 (s)  4 3.00 brt. (9) 4.89(OH) (w), 3.07 (s), 3.03 (s)  5 3.03 ddd (13, 9, 3.63 (w), 3.40 (s),3.00 (s) ~1)  6 3.63 brd. (11.8) 4.48 (OH) (m), 3.40 (s), 3.03 (w)3.40^(c) 4.48 (OH) (m), 3.63 (s), 3.03 (s)  1′ 3.78 ddd (15.1, 3.37 (s),2.10 (m), 1.93 (m) 9.4, ~1) 3.37^(c) 3.78 (s), 2.10 (m), 1.93 (m)  2′2.10 ddd (14.6, 3.78 (m), 3.37 (m), 1.93 (s) 8.5, 6) 1.93 ddd (14.6,3.78 (m), 3.37 (m), 2.10 (s) 7.3, ~1)  4′ 3.52 d (14.6) 3.33 (s)3.33^(c) 3.52 (s)  7′ 6.15 s (none)  9′ 2.77 dd (16.3, 4) 4.10 (s), 2.63(s) 2.63 d (16.3) 5.08 (vw), 4.10 (m), 2.77 (s) 10′ 4.10 brs. 4.92 (OH)(m), 2.77 (s), 2.63 (m) 11′ 5.08 s 6.95 (w), 6.72 (m), 2.63 (vw) c 6.50s 6.15 (w) e 6.95 brs. 6.72 (m), 6.15 (w), 5.08 (w) g 6.72 d (8.3) 6.95(w), 6.75 (s), 5.08 (m) h 6.75 d (8.3) 6.72 (s) ^(a)In DMSO-d₆.^(b)Intensities: s = strong, m = medium, w = weak. ^(c)Obscured by thewater signal.

Derivatization of Perseorajin

Hydrophobic derivatives of perseoranjin were prepared in order to extendthe potential color additive activity in foods containing significantamounts of fat. Derivatives were prepared by acylation of perseoranjinby alkali-catalyzed reaction with acyl chlorides. A summary of theexpected chemical modification is shown in Scheme 1, where R is analiphatic or aromatic chain.

In the reaction of scheme 1, R can be a 1) straight aliphatic chain 1-24carbons in length with 0-4 degrees of unsaturation; 2) branchedaliphatic chain 2-24 carbons in length with 0-4 degrees of unsaturation;3) phenyl functionality connected to the carbonyl carbon by an aliphaticchain 1-6 carbons in length with 0-3 degrees of unsaturation; 4)naphthyl functionality connected to the carbonyl carbon by an aliphaticchain 1-6 carbons in length with 0-3 degrees of unsaturation; 5) hydroxyphenyl functionality with 1-4 hydroxyl substitutions connected to thecarbonyl carbon by an aliphatic chain 1-6 carbons in length with 0-3degrees of unsaturation; or 6) hydroxy naphthyl functionality with 1-6hydroxyl substitutions connected to the carbonyl carbon by an aliphaticchain 1-6 carbons in length with 0-3 degrees of unsaturation.

Acetylation of Perseoranjin

AvoColor (1 mass equivalent) was suspended in 10 mass equivalents ofice-cold anhydrous dichloromethane. Triethylamine (43 mass equivalents)were added to the reaction. A catalytic amount of4-dimethylaminopyridine was added to the reaction. The reaction wasstirred on ice and acetyl chloride (16 mass equivalents) dissolved in 10mass equivalents of dichloromethane was added dropwise over 10 min. Thereaction was stirred overnight and allowed to return to roomtemperature. The reaction was stopped by addition of water. The reactionmixture was extracted with three times with dichloromethane. Thedichloromethane fraction was dried under vacuum to yield a red-brownsolid. The red-brown solid was readily soluble in acetone and ethylacetate, but not water. The red-brown product was solubilized in ethylacetate and extracted 3 times with 1 M HCl. The ethyl acetate fractionturned from red-brown to yellow-orange. The ethyl acetate fraction wasdried under vacuum to yield an orange solid. This solid is referred toas acetylated perseoranjin.

Benzoylation of Perseoranjin

AvoColor (1 mass equivalent) was suspended in 10 mass equivalents ofice-cold anhydrous dichloromethane. Triethylamine (48 mass equivalents)were added to the reaction. A catalytic amount of4-dimethylaminopyridine was added to the reaction. The reaction wasstirred on ice and acetyl chloride (40 mass equivalents) dissolved in 10mass equivalents of dichloromethane was added dropwise over 10 min. Thereaction was stirred overnight and allowed to return to roomtemperature. The reaction was stopped by addition of 1 M HCl. Thedichloromethane phase was yellow. The dichloromethane fraction wascollected and extracted three times with 1 M HCl. The dichloromethanephase was dried under vacuum yielding a yellow oil that is readilysoluble in ethyl acetate and dichloromethane but not water. This oil isreferred to as benzoylated perseoranjin.

The Avocolor compound can be isolated using the procedural flow chartdepicted in FIG. 75. Seeds of Persea Americana are washed in water andtheir size is reduced in two steps, first a coarse size reduction andsecond a fine size reduction. The product is then incubated for at least1 minute and up to a few days at a temperature of 0-40° C. Extraction ofperseoranjin is carried out using MeOH, EtOH or solvents with similarpolarities such as acetone or alcohol/water mixture. The liquid is thencollected by filtering the extracted product through a Whatman No. 4sieve to remove solids. A second filtration step, through a Whatman No.2 sieve, removes the starches. The impurities in the liquid are thenprecipitated by incubation for at least 24-48 hours at 4° C. Theprecipitate is removed through filtration or centrifugation and theliquid is collected. The liquid is undergoes sorption through resin,such as XAD-7. The resin is washed twice with water to remove thehydrophilic solutes. The perseoranjin is then eluted from the resinusing EtOH, MeOH, acetone, citric acid, acetic acid or any combinationthereof. The colorant is then concentrated by evaporation. If desired,the product can be dried through freeze drying or spray drying with anexcipient such as maltodextrin or a sugar.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1-17. (canceled)
 18. A stable compound selected from the groupconsisting of:

and salts thereof.
 19. The stable compound of claim 18, wherein thecompound is a hue selected from the group consisting of yellow, orange,and red.
 20. An edible material comprising a compound selected from thegroup consisting of

and

and salts thereof.
 21. The edible material of claim 20, wherein theedible material has a hue selected from the group consisting of orange,red, and yellow.
 22. A solution comprising a compound or salt of claim18 and at least one alcohol.
 23. The solution of claim 22, wherein thealcohol is selected from the group consisting of methanol and ethanol.24. The solution of claim 22, wherein the solution is substantiallylipid free.
 25. The solution of claim 22, wherein the solution issubstantially free of starch.
 26. The solution of claim 24, wherein thesolution is substantially free of starch.
 27. A method of impartingcolor to a substrate, comprising applying a solution of claim 22 to asubstrate.
 28. The method of claim 25, wherein the substrate is edible.29. The solution of claim 22, wherein the compound or salt is stable tolight.
 30. The solution of claim 22, wherein the compound or salt isstable to oxygen.
 31. A freeze-dried compound or salt of claim
 18. 32. Astable, isolated compound prepared by a process comprising: grinding aseed of Persea Americana to a slurry; incubating the slurry; extractinga compound with an alcohol to form a first mixture comprising acompound, starch, at least one water insoluble compound, and at leastone hydrophilic solute; removing the starch from the first mixture toform a second mixture; precipitating the at least one water insolublecompound to form a third mixture; adsorbing the third mixture to aresin; eluting a stable compound from the resin with alcohol; andevaporating the alcohol to form a stable, isolated compound.
 33. Asolution comprising the stable, isolated compound of claim 32 and atleast one alcohol.
 34. The solution of claim 33, wherein the solvent isselected from methanol, ethanol, acetone, citric acid, acetic acid, andcombinations thereof.
 35. The solution of claim 32, wherein the alcoholis selected from the group consisting of ethanol and methanol.
 36. Anacylated compound or salt of claim 18.